Bio


Herschlag studies mechanisms of biological catalysis by RNA and proteins, with the general goal of understanding how RNA and protein enzymes achieve their enormous rate enhancements and exquisite specificity. Mechanistic approaches are also being exploited to understand more complex biological processes, such as translation initiation, RNA chaperones, and the cellular behavior of RNA. Additional areas of investigation include catalysis of phosphoryl transfer by RNA and protein enzymes, model studies of phosphoryl transfer and hydrogen bond energetics, and RNA folding.

Academic Appointments


Administrative Appointments


  • Senior Associate Dean of Graduate Education and Postdoctoral Affairs, School of Medicine (2011 - Present)

Honors & Awards


  • Student Service Faculty Award, Biochemistry, Stanford School of Medicine (2012)
  • William Rose Award, ASBMB (2010)
  • Fellow, AAAS (2005)
  • Merit Award, NIH (2002)
  • Cope Scholar Award, ACS (2000)
  • Established Investigator, AHA (1998-2002)
  • Pfizer Award for Enzyme Chemistry, ACS (1997)
  • Fellowship in Science and Engineering, David and Lucile Packard (1995-2000)
  • Scholar, Searle (1993-1996)
  • Scholar in Biomedical Science, Lucille P. Markey (1990-1997)
  • Postdoctoral Fellowship, Helen Hay Whitney (Colorado; 1989-1990)
  • Fellowship, Gillette Foundation (Brandeis; 1986-1987)
  • Award for Excellence in Biochemistry, SUNY (1982)
  • Award in Biochemistry, American Institute of Chemists (SUNY; 1982)
  • Phi Beta Kappa, Michigan (1979)
  • Scholar, James B. Angell (Michigan; 1978, 1979)

Current Research and Scholarly Interests


Our research is aimed at understanding the chemical and physical behavior underlying biological macromolecules and systems, as these behaviors define the capabilities and limitations of biology. Toward this end we study folding and catalysis by RNA, as well as catalysis by protein enzymes. Comparing and contrasting the behavior of RNA and protein systems highlights the unique features of these biological macromolecules and helps elucidate principles of folding and catalysis that may be fundamental to both class of functional macromolecules. Toward these ends, many biochemical and biophysical techniques are employed, both within the lab and through multiple collaborations. Finally, we are also involved in collaborative work with Pat Brown's lab aimed at unraveling the global logic and organization of RNA processing and organization. More detailed descriptions are available from our lab home page.

2013-14 Courses


Graduate and Fellowship Programs


Journal Articles


  • Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sigala, P. A., Fafarman, A. T., Schwans, J. P., Fried, S. D., Fenn, T. D., Caaveiro, J. M., Pybus, B., Ringe, D., Petsko, G. A., Boxer, S. G., Herschlag, D. 2013; 110 (28): E2552-E2561

    Abstract

    Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol-Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations.

    View details for DOI 10.1073/pnas.1302191110

    View details for Web of Science ID 000321827000006

    View details for PubMedID 23798390

  • Use of anion-aromatic interactions to position the general base in the ketosteroid isomerase active site PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schwans, J. P., Sunden, F., Lassila, J. K., Gonzalez, A., Tsai, Y., Herschlag, D. 2013; 110 (28): 11308-11313

    Abstract

    Although the cation-pi pair, formed between a side chain or substrate cation and the negative electrostatic potential of a pi system on the face of an aromatic ring, has been widely discussed and has been shown to be important in protein structure and protein-ligand interactions, there has been little discussion of the potential structural and functional importance in proteins of the related anion-aromatic pair (i.e., interaction of a negatively charged group with the positive electrostatic potential on the ring edge of an aromatic group). We posited, based on prior structural information, that anion-aromatic interactions between the anionic Asp general base and Phe54 and Phe116 might be used instead of a hydrogen-bond network to position the general base in the active site of ketosteroid isomerase from Comamonas testosteroni as there are no neighboring hydrogen-bonding groups. We have tested the role of the Phe residues using site-directed mutagenesis, double-mutant cycles, and high-resolution X-ray crystallography. These results indicate a catalytic role of these Phe residues. Extensive analysis of the Protein Data Bank provides strong support for a catalytic role of these and other Phe residues in providing anion-aromatic interactions that position anionic general bases within enzyme active sites. Our results further reveal a potential selective advantage of Phe in certain situations, relative to more traditional hydrogen-bonding groups, because it can simultaneously aid in the binding of hydrophobic substrates and positioning of a neighboring general base.

    View details for DOI 10.1073/pnas.1206710110

    View details for Web of Science ID 000321827000037

    View details for PubMedID 23798413

  • Ground State Destabilization by Anionic Nucleophiles Contributes to the Activity of Phosphoryl Transfer Enzymes PLOS BIOLOGY Andrews, L. D., Fenn, T. D., Herschlag, D. 2013; 11 (7)

    Abstract

    Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 10(22)-fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed Pi affinity by orders of magnitude at pH 8.0. To allow precise and quantitative comparisons, the ionic form of bound Pi was determined from pH dependencies of the binding of Pi and tungstate, a Pi analog lacking titratable protons over the pH range of 5-11, and from the (31)P chemical shift of bound Pi. The results show that the Pi trianion binds with an exceptionally strong femtomolar affinity in the absence of Ser102, show that its binding is destabilized by ≥10(8)-fold by the Ser102 alkoxide, and provide direct evidence for ground state destabilization. Comparisons of X-ray crystal structures of AP with and without Ser102 reveal the same active site and Pi binding geometry upon removal of Ser102, suggesting that the destabilization does not result from a major structural rearrangement upon mutation of Ser102. Analogous Pi binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state destabilization mechanism. Our results have uncovered an important contribution of anionic nucleophiles to phosphoryl transfer catalysis via ground state electrostatic destabilization and an enormous capacity of the AP active site for specific and strong recognition of the phosphoryl group in the transition state.

    View details for DOI 10.1371/journal.pbio.1001599

    View details for Web of Science ID 000322592700003

    View details for PubMedID 23843744

  • Structural ensemble and microscopic elasticity of freely diffusing DNA by direct measurement of fluctuations. Proceedings of the National Academy of Sciences of the United States of America Shi, X., Herschlag, D., Harbury, P. A. 2013; 110 (16): E1444-51

    Abstract

    Precisely measuring the ensemble of conformers that a macromolecule populates in solution is highly challenging. Thus, it has been difficult to confirm or falsify the predictions of nanometer-scale dynamical modeling. Here, we apply an X-ray interferometry technique to probe the solution structure and fluctuations of B-form DNA on a length scale comparable to a protein-binding site. We determine an extensive set of intrahelix distance distributions between pairs of probes placed at distinct points on the surface of the DNA duplex. The distributions of measured distances reveal the nature and extent of the thermally driven mechanical deformations of the helix. We describe these deformations in terms of elastic constants, as is common for DNA and other polymers. The average solution structure and microscopic elasticity measured by X-ray interferometry are in striking agreement with values derived from DNA-protein crystal structures and measured by force spectroscopy, with one exception. The observed microscopic torsional rigidity of DNA is much lower than is measured by single-molecule twisting experiments, suggesting that torsional rigidity increases when DNA is stretched. Looking forward, molecular-level interferometry can provide a general tool for characterizing solution-phase structural ensembles.

    View details for DOI 10.1073/pnas.1218830110

    View details for PubMedID 23576725

  • Fundamental Challenges in Mechanistic Enzymology: Progress toward Understanding the Rate Enhancements of Enzymes BIOCHEMISTRY Herschlag, D., Natarajan, A. 2013; 52 (12): 2050-2067

    View details for DOI 10.1021/bi4000113

    View details for Web of Science ID 000316846800005

  • Ground State Destabilization from a Positioned General Base in the Ketosteroid Isomerase Active Site BIOCHEMISTRY Ruben, E. A., Schwans, J. P., Sonnett, M., Natarajan, A., Gonzalez, A., Tsai, Y., Herschlag, D. 2013; 52 (6): 1074-1081

    Abstract

    We compared the binding affinities of ground state analogues for bacterial ketosteroid isomerase (KSI) with a wild-type anionic Asp general base and with uncharged Asn and Ala in the general base position to provide a measure of potential ground state destabilization that could arise from the close juxtaposition of the anionic Asp and hydrophobic steroid in the reaction's Michaelis complex. The analogue binding affinity increased ~1 order of magnitude for the Asp38Asn mutation and ~2 orders of magnitude for the Asp38Ala mutation, relative to the affinity with Asp38, for KSI from two sources. The increased level of binding suggests that the abutment of a charged general base and a hydrophobic steroid is modestly destabilizing, relative to a standard state in water, and that this destabilization is relieved in the transition state and intermediate in which the charge on the general base has been neutralized because of proton abstraction. Stronger binding also arose from mutation of Pro39, the residue adjacent to the Asp general base, consistent with an ability of the Asp general base to now reorient to avoid the destabilizing interaction. Consistent with this model, the Pro mutants reduced or eliminated the increased level of binding upon replacement of Asp38 with Asn or Ala. These results, supported by additional structural observations, suggest that ground state destabilization from the negatively charged Asp38 general base provides a modest contribution to KSI catalysis. They also provide a clear illustration of the well-recognized concept that enzymes evolve for catalytic function and not, in general, to maximize ground state binding. This ground state destabilization mechanism may be common to the many enzymes with anionic side chains that deprotonate carbon acids.

    View details for DOI 10.1021/bi301348x

    View details for Web of Science ID 000315018600010

    View details for PubMedID 23311398

  • Fundamental Challenges in Mechanistic Enzymology: Progress toward Understanding the Rate Enhancements of Enzymes. Biochemistry Herschlag, D., Natarajan, A. 2013

    Abstract

    Enzymes are remarkable catalysts that lie at the heart of biology, accelerating chemical reactions to an astounding extent with extraordinary specificity. Enormous progress in understanding the chemical basis of enzymatic transformations and the basic mechanisms underlying rate enhancements over the past decades is apparent. Nevertheless, it has been difficult to achieve a quantitative understanding of how the underlying mechanisms account for the energetics of catalysis, because of the complexity of enzyme systems and the absence of underlying energetic additivity. We review case studies from our own work that illustrate the power of precisely defined and clearly articulated questions when dealing with such complex and multifaceted systems, and we also use this approach to evaluate our current ability to design enzymes. We close by highlighting a series of questions that help frame some of what remains to be understood, and we encourage the reader to define additional questions and directions that will deepen and broaden our understanding of enzymes and their catalysis.

    View details for PubMedID 23488725

  • Salt dependence of the radius of gyration and flexibility of single-stranded DNA in solution probed by small-angle x-ray scattering PHYSICAL REVIEW E Sim, A. Y., Lipfert, J., Herschlag, D., Doniach, S. 2012; 86 (2)

    Abstract

    Short single-stranded nucleic acids are ubiquitous in biological processes; understanding their physical properties provides insights to nucleic acid folding and dynamics. We used small-angle x-ray scattering to study 8-100 residue homopolymeric single-stranded DNAs in solution, without external forces or labeling probes. Poly-T's structural ensemble changes with increasing ionic strength in a manner consistent with a polyelectrolyte persistence length theory that accounts for molecular flexibility. For any number of residues, poly-A is consistently more elongated than poly-T, likely due to the tendency of A residues to form stronger base-stacking interactions than T residues.

    View details for DOI 10.1103/PhysRevE.86.021901

    View details for Web of Science ID 000307277300008

    View details for PubMedID 23005779

  • Metal-ion rescue revisited: Biochemical detection of site-bound metal ions important for RNA folding RNA-A PUBLICATION OF THE RNA SOCIETY Frederiksen, J. K., Li, N., Das, R., Herschlag, D., Piccirilli, J. A. 2012; 18 (6): 1123-1141

    Abstract

    Within the three-dimensional architectures of RNA molecules, divalent metal ions populate specific locations, shedding their water molecules to form chelates. These interactions help the RNA adopt and maintain specific conformations and frequently make essential contributions to function. Defining the locations of these site-bound metal ions remains challenging despite the growing database of RNA structures. Metal-ion rescue experiments have provided a powerful approach to identify and distinguish catalytic metal ions within RNA active sites, but the ability of such experiments to identify metal ions that contribute to tertiary structure acquisition and structural stability is less developed and has been challenged. Herein, we use the well-defined P4-P6 RNA domain of the Tetrahymena group I intron to reevaluate prior evidence against the discriminatory power of metal-ion rescue experiments and to advance thermodynamic descriptions necessary for interpreting these experiments. The approach successfully identifies ligands within the RNA that occupy the inner coordination sphere of divalent metal ions and distinguishes them from ligands that occupy the outer coordination sphere. Our results underscore the importance of obtaining complete folding isotherms and establishing and evaluating thermodynamic models in order to draw conclusions from metal-ion rescue experiments. These results establish metal-ion rescue as a rigorous tool for identifying and dissecting energetically important metal-ion interactions in RNAs that are noncatalytic but critical for RNA tertiary structure.

    View details for DOI 10.1261/rna.028738.111

    View details for Web of Science ID 000304423000003

    View details for PubMedID 22539523

  • Exploring purine N7 interactions via atomic mutagenesis: The group I ribozyme as a case study RNA-A PUBLICATION OF THE RNA SOCIETY Forconi, M., Benz-Moy, T., Gleitsman, K. R., Ruben, E., Metz, C., Herschlag, D. 2012; 18 (6): 1222-1229

    Abstract

    Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure-function relationships analogous to those seen for protein ligands and provided strong evidence for a well-formed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by ~20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes.

    View details for DOI 10.1261/rna.031567.111

    View details for Web of Science ID 000304423000011

    View details for PubMedID 22543863

  • Robust design and optimization of retroaldol enzymes PROTEIN SCIENCE Althoff, E. A., Wang, L., Jiang, L., Giger, L., Lassila, J. K., Wang, Z., Smith, M., Hari, S., Kast, P., Herschlag, D., Hilvert, D., Baker, D. 2012; 21 (5): 717-726

    Abstract

    Enzyme catalysts of a retroaldol reaction have been generated by computational design using a motif that combines a lysine in a nonpolar environment with water-mediated stabilization of the carbinolamine hydroxyl and ?-hydroxyl groups. Here, we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site-directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride-reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited.

    View details for DOI 10.1002/pro.2059

    View details for Web of Science ID 000302620600012

    View details for PubMedID 22407837

  • Electrostatics of Nucleic Acid Folding under Conformational Constraint JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Anthony, P. C., Sim, A. Y., Chu, V. B., Doniach, S., Block, S. M., Herschlag, D. 2012; 134 (10): 4607-4614

    Abstract

    RNA folding is enabled by interactions between the nucleic acid and its ion atmosphere, the mobile sheath of aqueous ions that surrounds and stabilizes it. Understanding the ion atmosphere requires the interplay of experiment and theory. However, even an apparently simple experiment to probe the ion atmosphere, measuring the dependence of DNA duplex stability upon ion concentration and identity, suffers from substantial complexity, because the unfolded ensemble contains many conformational states that are difficult to treat accurately with theory. To minimize this limitation, we measured the unfolding equilibrium of a DNA hairpin using a single-molecule optical trapping assay, in which the unfolded state is constrained to a limited set of elongated conformations. The unfolding free energy increased linearly with the logarithm of monovalent cation concentration for several cations, such that smaller cations tended to favor the folded state. Mg(2+) stabilized the hairpin much more effectively at low concentrations than did any of the monovalent cations. Poisson-Boltzmann theory captured trends in hairpin stability measured for the monovalent cation titrations with reasonable accuracy, but failed to do so for the Mg(2+) titrations. This finding is consistent with previous work, suggesting that Poisson-Boltzmann and other mean-field theories fail for higher valency cations where ion-ion correlation effects may become significant. The high-resolution data herein, because of the straightforward nature of both the folded and the unfolded states, should serve as benchmarks for the development of more accurate electrostatic theories that will be needed for a more quantitative and predictive understanding of nucleic acid folding.

    View details for DOI 10.1021/ja208466h

    View details for Web of Science ID 000301990600036

    View details for PubMedID 22369617

  • Single Molecule Analysis Research Tool (SMART): An Integrated Approach for Analyzing Single Molecule Data PLOS ONE Greenfeld, M., Pavlichin, D. S., Mabuchi, H., Herschlag, D. 2012; 7 (2)

    Abstract

    Single molecule studies have expanded rapidly over the past decade and have the ability to provide an unprecedented level of understanding of biological systems. A common challenge upon introduction of novel, data-rich approaches is the management, processing, and analysis of the complex data sets that are generated. We provide a standardized approach for analyzing these data in the freely available software package SMART: Single Molecule Analysis Research Tool. SMART provides a format for organizing and easily accessing single molecule data, a general hidden Markov modeling algorithm for fitting an array of possible models specified by the user, a standardized data structure and graphical user interfaces to streamline the analysis and visualization of data. This approach guides experimental design, facilitating acquisition of the maximal information from single molecule experiments. SMART also provides a standardized format to allow dissemination of single molecule data and transparency in the analysis of reported data.

    View details for DOI 10.1371/journal.pone.0030024

    View details for Web of Science ID 000302871500004

    View details for PubMedID 22363412

  • Thermodynamic evidence for negative charge stabilization by a catalytic metal ion within an RNA active site. ACS chemical biology Sengupta, R. N., Herschlag, D., Piccirilli, J. A. 2012; 7 (2): 294-299

    Abstract

    Protein and RNA enzymes that catalyze phosphoryl transfer reactions frequently contain active site metal ions that interact with the nucleophile and leaving group. Mechanistic models generally hinge upon the assumption that the metal ions stabilize negative charge buildup along the reaction coordinate. However, experimental data that test this assumption directly remain difficult to acquire. We have used an RNA substrate bearing a 3'-thiol group to investigate the energetics of a metal ion interaction directly relevant to transition state stabilization in the Tetrahymena group I ribozyme reaction. Our results show that this interaction lowers the pK(a) of the 3'-thiol by 2.6 units, stabilizing the bound 3'-thiolate by 3.6 kcal/mol. These data, combined with prior studies, provide strong evidence that this metal ion interaction facilitates the forward reaction by stabilization of negative charge buildup on the leaving group 3'-oxygen and facilitates the reverse reaction by deprotonation and activation of the nucleophilic 3'-hydroxyl group.

    View details for DOI 10.1021/cb200202q

    View details for PubMedID 22029738

  • Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fafarman, A. T., Sigala, P. A., Schwans, J. P., Fenn, T. D., Herschlag, D., Boxer, S. G. 2012; 109 (6): E299-E308

    Abstract

    Understanding the electrostatic forces and features within highly heterogeneous, anisotropic, and chemically complex enzyme active sites and their connection to biological catalysis remains a longstanding challenge, in part due to the paucity of incisive experimental probes of electrostatic properties within proteins. To quantitatively assess the landscape of electrostatic fields at discrete locations and orientations within an enzyme active site, we have incorporated site-specific thiocyanate vibrational probes into multiple positions within bacterial ketosteroid isomerase. A battery of X-ray crystallographic, vibrational Stark spectroscopy, and NMR studies revealed electrostatic field heterogeneity of 8 MV/cm between active site probe locations and widely differing sensitivities of discrete probes to common electrostatic perturbations from mutation, ligand binding, and pH changes. Electrostatic calculations based on active site ionization states assigned by literature precedent and computational pK(a) prediction were unable to quantitatively account for the observed vibrational band shifts. However, electrostatic models of the D40N mutant gave qualitative agreement with the observed vibrational effects when an unusual ionization of an active site tyrosine with a pK(a) near 7 was included. UV-absorbance and (13)C NMR experiments confirmed the presence of a tyrosinate in the active site, in agreement with electrostatic models. This work provides the most direct measure of the heterogeneous and anisotropic nature of the electrostatic environment within an enzyme active site, and these measurements provide incisive benchmarks for further developing accurate computational models and a foundation for future tests of electrostatics in enzymatic catalysis.

    View details for DOI 10.1073/pnas.1111566109

    View details for Web of Science ID 000299925000004

    View details for PubMedID 22308339

  • A Role for a Single-Stranded Junction in RNA Binding and Specificity by the Tetrahymena Group I Ribozyme JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Shi, X., Solomatin, S. V., Herschlag, D. 2012; 134 (4): 1910-1913

    Abstract

    We have investigated the role of a single-stranded RNA junction, J1/2, that connects the substrate-containing P1 duplex to the remainder of the Tetrahymena group I ribozyme. Single-turnover kinetics, fluorescence anisotropy, and single-molecule fluorescence resonance energy transfer studies of a series of J1/2 mutants were used to probe the sequence dependence of the catalytic activity, the P1 dynamics, and the thermodynamics of docking of the P1 duplex into the ribozyme's catalytic core. We found that A29, the center A of three adenosine residues in J1/2, contributes 2 orders of magnitude to the overall ribozyme activity, and double-mutant cycles suggested that J1/2 stabilizes the docked state of P1 over the undocked state via a tertiary interaction involving A29 and the first base pair in helix P2 of the ribozyme, A31·U56. Comparative sequence analysis of this group I intron subclass suggests that the A29 interaction sets one end of a molecular ruler whose other end specifies the 5'-splice site and that this molecular ruler is conserved among a subclass of group I introns related to the Tetrahymena intron. Our results reveal substantial functional effects from a seemingly simple single-stranded RNA junction and suggest that junction sequences may evolve rapidly to provide important interactions in functional RNAs.

    View details for DOI 10.1021/ja2083575

    View details for Web of Science ID 000301084600005

    View details for PubMedID 22220837

  • Thermodynamic Evidence for Negative Charge Stabilization by a Catalytic Metal Ion within an RNA Active Site ACS CHEMICAL BIOLOGY Sengupta, R. N., Herschlag, D., Piccirilli, J. A. 2012; 7 (2): 293-298

    View details for DOI 10.1021/cb200202q

    View details for Web of Science ID 000300339600007

  • High-Resolution Analysis of Zn2+ Coordination in the Alkaline Phosphatase Superfamily by EXAFS and X-ray Crystallography JOURNAL OF MOLECULAR BIOLOGY Bobyr, E., Lassila, J. K., Wiersma-Koch, H. I., Fenn, T. D., Lee, J. J., Nikolic-Hughes, I., Hodgson, K. O., Rees, D. C., Hedman, B., Herschlag, D. 2012; 415 (1): 102-117

    Abstract

    Comparisons among evolutionarily related enzymes offer opportunities to reveal how structural differences produce different catalytic activities. Two structurally related enzymes, Escherichia coli alkaline phosphatase (AP) and Xanthomonas axonopodis nucleotide pyrophosphatase/phosphodiesterase (NPP), have nearly identical binuclear Zn(2+) catalytic centers but show tremendous differential specificity for hydrolysis of phosphate monoesters or phosphate diesters. To determine if there are differences in Zn(2+) coordination in the two enzymes that might contribute to catalytic specificity, we analyzed both x-ray absorption spectroscopic and x-ray crystallographic data. We report a 1.29-Å crystal structure of AP with bound phosphate, allowing evaluation of interactions at the AP metal site with high resolution. To make systematic comparisons between AP and NPP, we measured zinc extended x-ray absorption fine structure for AP and NPP in the free-enzyme forms, with AMP and inorganic phosphate ground-state analogs and with vanadate transition-state analogs. These studies yielded average zinc-ligand distances in AP and NPP free-enzyme forms and ground-state analog forms that were identical within error, suggesting little difference in metal ion coordination among these forms. Upon binding of vanadate to both enzymes, small increases in average metal-ligand distances were observed, consistent with an increased coordination number. Slightly longer increases were observed in NPP relative to AP, which could arise from subtle rearrangements of the active site or differences in the geometry of the bound vanadyl species. Overall, the results suggest that the binuclear Zn(2+) catalytic site remains very similar between AP and NPP during the course of a reaction cycle.

    View details for DOI 10.1016/j.jmb.2011.10.040

    View details for Web of Science ID 000299866000010

    View details for PubMedID 22056344

  • Evaluating the Catalytic Contribution from the Oxyanion Hole in Ketosteroid Isomerase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Schwans, J. P., Sunden, F., Gonzalez, A., Tsai, Y., Herschlag, D. 2011; 133 (50): 20052-20055

    Abstract

    Prior site-directed mutagenesis studies in bacterial ketosteroid isomerase (KSI) reported that substitution of both oxyanion hole hydrogen bond donors gives a 10(5)- to 10(8)-fold rate reduction, suggesting that the oxyanion hole may provide the major contribution to KSI catalysis. But these seemingly conservative mutations replaced the oxyanion hole hydrogen bond donors with hydrophobic side chains that could lead to suboptimal solvation of the incipient oxyanion in the mutants, thereby potentially exaggerating the apparent energetic benefit of the hydrogen bonds relative to water-mediated hydrogen bonds in solution. We determined the functional and structural consequences of substituting the oxyanion hole hydrogen bond donors and several residues surrounding the oxyanion hole with smaller residues in an attempt to create a local site that would provide interactions more analogous to those in aqueous solution. These more drastic mutations created an active-site cavity estimated to be ~650 Å(3) and sufficient for occupancy by 15-17 water molecules and led to a rate decrease of only ~10(3)-fold for KSI from two different species, a much smaller effect than that observed from more traditional conservative mutations. The results underscore the strong context dependence of hydrogen bond energetics and suggest that the oxyanion hole provides an important, but moderate, catalytic contribution relative to the interactions in the corresponding solution reaction.

    View details for DOI 10.1021/ja208050t

    View details for Web of Science ID 000298713600008

    View details for PubMedID 22053826

  • Structure-Function Analysis from the Outside In: Long-Range Tertiary Contacts in RNA Exhibit Distinct Catalytic Roles BIOCHEMISTRY Benz-Moy, T. L., Herschag, D. 2011; 50 (40): 8733-8755

    Abstract

    The conserved catalytic core of the Tetrahymena group I ribozyme is encircled by peripheral elements. We have conducted a detailed structure-function study of the five long-range tertiary contacts that fasten these distal elements together. Mutational ablation of each of the tertiary contacts destabilizes the folded ribozyme, indicating a role of the peripheral elements in overall stability. Once folded, three of the five tertiary contact mutants exhibit defects in overall catalysis that range from 20- to 100-fold. These and the subsequent results indicate that the structural ring of peripheral elements does not act as a unitary element; rather, individual connections have distinct roles as further revealed by kinetic and thermodynamic dissection of the individual reaction steps. Ablation of P14 or the metal ion core/metal ion core receptor (MC/MCR) destabilizes docking of the substrate-containing P1 helix into tertiary interactions with the ribozyme's conserved core. In contrast, ablation of the L9/P5 contact weakens binding of the guanosine nucleophile by slowing its association, without affecting P1 docking. The P13 and tetraloop/tetraloop receptor (TL/TLR) mutations had little functional effect and small, local structural changes, as revealed by hydroxyl radical footprinting, whereas the P14, MC/MCR, and L9/P5 mutants show structural changes distal from the mutation site. These changes extended into regions of the catalytic core involved in docking or guanosine binding. Thus, distinct allosteric pathways couple the long-range tertiary contacts to functional sites within the conserved core. This modular functional specialization may represent a fundamental strategy in RNA structure-function interrelationships.

    View details for DOI 10.1021/bi2008245

    View details for Web of Science ID 000295487700022

    View details for PubMedID 21815635

  • 2 '-Fluoro Substituents Can Mimic Native 2 '-Hydroxyls within Structured RNA CHEMISTRY & BIOLOGY Forconi, M., Schwans, J. P., Porecha, R. H., Sengupta, R. N., Piccirilli, J. A., Herschlag, D. 2011; 18 (8): 949-954

    Abstract

    The ability of fluorine in a C-F bond to act as a hydrogen bond acceptor is controversial. To test such ability in complex RNA macromolecules, we have replaced native 2'-OH groups with 2'-F and 2'-H groups in two related systems, the Tetrahymena group I ribozyme and the ?C209 P4-P6 RNA domain. In three cases the introduced 2'-F mimics the native 2'-OH group, suggesting that the fluorine atom can accept a hydrogen bond. In each of these cases the native hydroxyl group interacts with a purine exocyclic amine. Our results give insight about the properties of organofluorine and suggest a possible general biochemical signature for tertiary interactions between 2'-hydroxyl groups and exocyclic amino groups within RNA.

    View details for DOI 10.1016/j.chembiol.2011.07.014

    View details for Web of Science ID 000295203200005

    View details for PubMedID 21867910

  • Isotope-Edited FTIR of Alkaline Phosphatase Resolves Paradoxical Ligand Binding Properties and Suggests a Role for Ground-State Destabilization JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Andrews, L. D., Deng, H., Herschlag, D. 2011; 133 (30): 11621-11631

    Abstract

    Escherichia coli alkaline phosphatase (AP) can hydrolyze a variety of chemically diverse phosphate monoesters while making contacts solely to the transferred phosphoryl group and its incoming and outgoing atoms. Strong interactions between AP and the transferred phosphoryl group are not present in the ground state despite the apparent similarity of the phosphoryl group in the ground and transition states. Such modest ground-state affinity is required to curtail substrate saturation and product inhibition and to allow efficient catalysis. To investigate how AP achieves limited affinity for its ground state, we first compared binding affinities of several related AP ligands. This comparison revealed a paradox: AP has a much stronger affinity for inorganic phosphate (P(i)) than for related compounds that are similar to P(i) geometrically and in overall charge but lack a transferable proton. We postulated that the P(i) proton could play an important role via transfer to the nearby anion, the active site serine nucleophile (Ser102), resulting in the attenuation of electrostatic repulsion between bound P(i) and the Ser102 oxyanion and the binding of P(i) in its trianionic form adjacent to a now neutral Ser residue. To test this model, isotope-edited Fourier transform infrared (FTIR) spectroscopy was used to investigate the ionic structure of AP-bound P(i). The FTIR results indicate that the P(i) trianion is bound and, in conjunction with previous studies of pH-dependent P(i) binding and other results, suggest that P(i) dianion transfers its proton to the Ser102 anion of AP. This internal proton-transfer results in stronger P(i) binding presumably because the additional negative charge on the trianionic P(i) allows stronger electrostatic interactions within the AP active site and because the electrostatic repulsion between bound P(i) and anionic Ser102 is eliminated when the transferred P(i) proton neutralizes Ser102. Indeed, when Ser102 is neutralized the P(i) trianion binds AP with a calculated K(d) of ?290 fM. These results suggest that electrostatic repulsion between Ser102 and negatively charged phosphate ester substrates contributes to catalysis by the preferential destabilization of the reaction's E·S ground state.

    View details for DOI 10.1021/ja203370b

    View details for Web of Science ID 000293872800042

    View details for PubMedID 21692505

  • Removal of Covalent Heterogeneity Reveals Simple Folding Behavior for P4-P6 RNA JOURNAL OF BIOLOGICAL CHEMISTRY Greenfeld, M., Solomatin, S. V., Herschlag, D. 2011; 286 (22): 19872-19879

    Abstract

    RNA folding landscapes have been described alternately as simple and as complex. The limited diversity of RNA residues and the ability of RNA to form stable secondary structures prior to adoption of a tertiary structure would appear to simplify folding relative to proteins. Nevertheless, there is considerable evidence for long-lived misfolded RNA states, and these observations have suggested rugged energy landscapes. Recently, single molecule fluorescence resonance energy transfer (smFRET) studies have exposed heterogeneity in many RNAs, consistent with deeply furrowed rugged landscapes. We turned to an RNA of intermediate complexity, the P4-P6 domain from the Tetrahymena group I intron, to address basic questions in RNA folding. P4-P6 exhibited long-lived heterogeneity in smFRET experiments, but the inability to observe exchange in the behavior of individual molecules led us to probe whether there was a non-conformational origin to this heterogeneity. We determined that routine protocols in RNA preparation and purification, including UV shadowing and heat annealing, cause covalent modifications that alter folding behavior. By taking measures to avoid these treatments and by purifying away damaged P4-P6 molecules, we obtained a population of P4-P6 that gave near-uniform behavior in single molecule studies. Thus, the folding landscape of P4-P6 lacks multiple deep furrows that would trap different P4-P6 molecules in different conformations and contrasts with the molecular heterogeneity that has been seen in many smFRET studies of structured RNAs. The simplicity of P4-P6 allowed us to reliably determine the thermodynamic and kinetic effects of metal ions on folding and to now begin to build more detailed models for RNA folding behavior.

    View details for DOI 10.1074/jbc.M111.235465

    View details for Web of Science ID 000291027700067

    View details for PubMedID 21478155

  • Implications of molecular heterogeneity for the cooperativity of biological macromolecules NATURE STRUCTURAL & MOLECULAR BIOLOGY Solomatin, S. V., Greenfeld, M., Herschlag, D. 2011; 18 (6): 732-734

    Abstract

    Cooperativity, a universal property of biological macromolecules, is typically characterized by a Hill slope, which can provide fundamental information about binding sites and interactions. We demonstrate, through simulations and single-molecule FRET (smFRET) experiments, that molecular heterogeneity lowers bulk cooperativity from the intrinsic value for the individual molecules. As heterogeneity is common in smFRET experiments, appreciation of its influence on fundamental measures of cooperativity is critical for deriving accurate molecular models.

    View details for DOI 10.1038/nsmb.2052

    View details for Web of Science ID 000291308000016

    View details for PubMedID 21572445

  • Tightening of Active Site Interactions En Route to the Transition State Revealed by Single-Atom Substitution in the Guanosine-Binding Site of the Tetrahymena Group I Ribozyme JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Forconi, M., Porecha, R. H., Piccirilli, J. A., Herschlag, D. 2011; 133 (20): 7791-7800

    Abstract

    Protein enzymes establish intricate networks of interactions to bind and position substrates and catalytic groups within active sites, enabling stabilization of the chemical transition state. Crystal structures of several RNA enzymes also suggest extensive interaction networks, despite RNA's structural limitations, but there is little information on the functional and the energetic properties of these inferred networks. We used double mutant cycles and presteady-state kinetic analyses to probe the putative interaction between the exocyclic amino group of the guanosine nucleophile and the N7 atom of residue G264 of the Tetrahymena group I ribozyme. As expected, the results supported the presence of this interaction, but remarkably, the energetic penalty for introducing a CH group at the 7-position of residue G264 accumulates as the reaction proceeds toward the chemical transition state to a total of 6.2 kcal/mol. Functional tests of neighboring interactions revealed that the presence of the CH group compromises multiple contacts within the interaction network that encompass the reactive elements, apparently forcing the nucleophile to bind and attack from an altered, suboptimal orientation. The energetic consequences of this indirect disruption of neighboring interactions as the reaction proceeds demonstrate that linkage between binding interactions and catalysis hinges critically on the precise structural integrity of a network of interacting groups.

    View details for DOI 10.1021/ja111316y

    View details for Web of Science ID 000291580400035

    View details for PubMedID 21539364

  • Identification of RNA recognition elements in the Saccharomyces cerevisiae transcriptome NUCLEIC ACIDS RESEARCH Riordan, D. P., Herschlag, D., Brown, P. O. 2011; 39 (4): 1501-1509

    Abstract

    Post-transcriptional regulation of gene expression, including mRNA localization, translation and decay, is ubiquitous yet still largely unexplored. How is the post-transcriptional regulatory program of each mRNA encoded in its sequence? Hundreds of specific RNA-binding proteins (RBPs) appear to play roles in mediating the post-transcriptional regulatory program, akin to the roles of specific DNA-binding proteins in transcription. As a step toward decoding the regulatory programs encoded in each mRNA, we focused on specific mRNA-protein interactions. We computationally analyzed the sequences of Saccharomyces cerevisiae mRNAs bound in vivo by 29 specific RBPs, identifying eight novel candidate motifs and confirming or extending six earlier reported recognition elements. Biochemical selections for RNA sequences selectively recognized by 12 yeast RBPs yielded novel motifs bound by Pin4, Nsr1, Hrb1, Gbp2, Sgn1 and Mrn1, and recovered the known recognition elements for Puf3, She2, Vts1 and Whi3. Most of the RNA elements we uncovered were associated with coherent mRNA expression changes and were significantly conserved in related yeasts, supporting their functional importance and suggesting that the corresponding RNA-protein interactions are evolutionarily conserved.

    View details for DOI 10.1093/nar/gkq920

    View details for Web of Science ID 000288019400036

    View details for PubMedID 20959291

  • Biological Phosphoryl-Transfer Reactions: Understanding Mechanism and Catalysis ANNUAL REVIEW OF BIOCHEMISTRY, VOL 80 Lassila, J. K., Zalatan, J. G., Herschlag, D. 2011; 80: 669-702

    Abstract

    Phosphoryl-transfer reactions are central to biology. These reactions also have some of the slowest nonenzymatic rates and thus require enormous rate accelerations from biological catalysts. Despite the central importance of phosphoryl transfer and the fascinating catalytic challenges it presents, substantial confusion persists about the properties of these reactions. This confusion exists despite decades of research on the chemical mechanisms underlying these reactions. Here we review phosphoryl-transfer reactions with the goal of providing the reader with the conceptual and experimental background to understand this body of work, to evaluate new results and proposals, and to apply this understanding to enzymes. We describe likely resolutions to some controversies, while emphasizing the limits of our current approaches and understanding. We apply this understanding to enzyme-catalyzed phosphoryl transfer and provide illustrative examples of how this mechanistic background can guide and deepen our understanding of enzymes and their mechanisms of action. Finally, we present important future challenges for this field.

    View details for DOI 10.1146/annurev-biochem-060409-092741

    View details for Web of Science ID 000292895600028

    View details for PubMedID 21513457

  • Hydrogen Bonding in the Active Site of Ketosteroid Isomerase: Electronic Inductive Effects and Hydrogen Bond Coupling BIOCHEMISTRY Hanoian, P., Sigala, P. A., Herschlag, D., Hammes-Schiffer, S. 2010; 49 (48): 10339-10348

    Abstract

    Computational studies are performed to analyze the physical properties of hydrogen bonds donated by Tyr16 and Asp103 to a series of substituted phenolate inhibitors bound in the active site of ketosteroid isomerase (KSI). As the solution pK(a) of the phenolate increases, these hydrogen bond distances decrease, the associated nuclear magnetic resonance (NMR) chemical shifts increase, and the fraction of protonated inhibitor increases, in agreement with prior experiments. The quantum mechanical/molecular mechanical calculations provide insight into the electronic inductive effects along the hydrogen bonding network that includes Tyr16, Tyr57, and Tyr32, as well as insight into hydrogen bond coupling in the active site. The calculations predict that the most-downfield NMR chemical shift observed experimentally corresponds to the Tyr16-phenolate hydrogen bond and that Tyr16 is the proton donor when a bound naphtholate inhibitor is observed to be protonated in electronic absorption experiments. According to these calculations, the electronic inductive effects along the hydrogen bonding network of tyrosines cause the Tyr16 hydroxyl to be more acidic than the Asp103 carboxylic acid moiety, which is immersed in a relatively nonpolar environment. When one of the distal tyrosine residues in the network is mutated to phenylalanine, thereby diminishing this inductive effect, the Tyr16-phenolate hydrogen bond becomes longer and the Asp103-phenolate hydrogen bond shorter, as observed in NMR experiments. Furthermore, the calculations suggest that the differences in the experimental NMR data and electronic absorption spectra for pKSI and tKSI, two homologous bacterial forms of the enzyme, are due predominantly to the third tyrosine that is present in the hydrogen bonding network of pKSI but not tKSI. These studies also provide experimentally testable predictions about the impact of mutating the distal tyrosine residues in this hydrogen bonding network on the NMR chemical shifts and electronic absorption spectra.

    View details for DOI 10.1021/bi101428e

    View details for Web of Science ID 000284717600010

    View details for PubMedID 21049962

  • Decomposition of Vibrational Shifts of Nitriles into Electrostatic and Hydrogen-Bonding Effects JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Fafarman, A. T., Sigala, P. A., Herschlag, D., Boxer, S. G. 2010; 132 (37): 12811-12813

    Abstract

    Infrared (IR) band shifts of isolated vibrational transitions can serve as quantitative and directional probes of local electrostatic fields, due to the vibrational Stark effect. However, departures from the Stark model can arise when the probe participates in specific, chemical interactions, such as direct hydrogen bonding. We present a method to identify and correct for these departures based on comparison of (13)C NMR chemical shifts and IR frequencies each calibrated in turn by a solvatochromic model. We demonstrate how the tandem use of these experimental observables can be applied to a thiocyanate-modified protein, ketosteroid isomerase, and show, by comparison to structural models, that changes in electrostatic field can be measured within the complex protein environment even in the background of direct hydrogen bonding to the probe.

    View details for DOI 10.1021/ja104573b

    View details for Web of Science ID 000282013700018

    View details for PubMedID 20806897

  • Multiple Unfolding Events during Native Folding of the Tetrahymena Group I Ribozyme JOURNAL OF MOLECULAR BIOLOGY Wan, Y., Suh, H., Russell, R., Herschlag, D. 2010; 400 (5): 1067-1077

    Abstract

    Despite the ubiquitous nature of misfolded intermediates in RNA folding, little is known about their physical properties or the folding transitions that allow them to continue folding productively. Folding of the Tetrahymena group I ribozyme includes sequential accumulation of two intermediates, termed I(trap) and misfolded (M). Here, we probe the structure and folding transition of I(trap) and compare them to those of M. Hydroxyl radical and dimethyl sulfate footprinting show that both I(trap) and M are extensively structured and crudely resemble the native RNA. However, regions of the core P3-P8 domain are more exposed to solvent in I(trap) than in M. I(trap) rearranges to continue folding nearly 1000-fold faster than M, and urea accelerates folding of I(trap) much less than M. Thus, the rate-limiting transition from I(trap) requires a smaller increase in exposed surface. Mutations that disrupt peripheral tertiary contacts give large and nearly uniform increases in re-folding of M, whereas the same mutations give at most modest increases in folding from I(trap). Intriguingly, mutations within the peripheral element P5abc give 5- to 10-fold accelerations in escape from I(trap), whereas ablation of P13, which lies on the opposite surface in the native structure, near the P3-P8 domain, has no effect. Thus, the unfolding required from I(trap) appears to be local, whereas the unfolding of M appears to be global. Further, the modest effects from several mutations suggest that there are multiple pathways for escape from I(trap) and that escape is aided by loosening nearby native structural constraints, presumably to facilitate local movements of nucleotides or segments that have not formed native contacts. Overall, these and prior results suggest a model in which the global architecture and peripheral interactions of the RNA are achieved relatively early in folding. Multiple folding and re-folding events occur on the predominant pathway to the native state, with increasing native core interactions and cooperativity as folding progresses.

    View details for DOI 10.1016/j.jmb.2010.06.010

    View details for Web of Science ID 000280533200011

    View details for PubMedID 20541557

  • Dissecting electrostatic screening, specific ion binding, and ligand binding in an energetic model for glycine riboswitch folding RNA-A PUBLICATION OF THE RNA SOCIETY Lipfert, J., Sim, A. Y., Herschlag, D., Doniach, S. 2010; 16 (4): 708-719

    Abstract

    Riboswitches are gene-regulating RNAs that are usually found in the 5'-untranslated regions of messenger RNA. As the sugar-phosphate backbone of RNA is highly negatively charged, the folding and ligand-binding interactions of riboswitches are strongly dependent on the presence of cations. Using small angle X-ray scattering (SAXS) and hydroxyl radical footprinting, we examined the cation dependence of the different folding stages of the glycine-binding riboswitch from Vibrio cholerae. We found that the partial folding of the tandem aptamer of this riboswitch in the absence of glycine is supported by all tested mono- and divalent ions, suggesting that this transition is mediated by nonspecific electrostatic screening. Poisson-Boltzmann calculations using SAXS-derived low-resolution structural models allowed us to perform an energetic dissection of this process. The results showed that a model with a constant favorable contribution to folding that is opposed by an unfavorable electrostatic term that varies with ion concentration and valency provides a reasonable quantitative description of the observed folding behavior. Glycine binding, on the other hand, requires specific divalent ions binding based on the observation that Mg(2+), Ca(2+), and Mn(2+) facilitated glycine binding, whereas other divalent cations did not. The results provide a case study of how ion-dependent electrostatic relaxation, specific ion binding, and ligand binding can be coupled to shape the energetic landscape of a riboswitch and can begin to be quantitatively dissected.

    View details for DOI 10.1261/rna.1985110

    View details for Web of Science ID 000275951000006

    View details for PubMedID 20194520

  • A Rearrangement of the Guanosine-Binding Site Establishes an Extended Network of Functional Interactions in the Tetrahymena Group I Ribozyme Active Site BIOCHEMISTRY Forconi, M., Sengupta, R. N., Piccirilli, J. A., Herschlag, D. 2010; 49 (12): 2753-2762

    Abstract

    Protein enzymes appear to use extensive packing and hydrogen bonding interactions to precisely position catalytic groups within active sites. Because of their inherent backbone flexibility and limited side chain repertoire, RNA enzymes face additional challenges relative to proteins in precisely positioning substrates and catalytic groups. Here, we use the group I ribozyme to probe the existence, establishment, and functional consequences of an extended network of interactions in an RNA active site. The group I ribozyme catalyzes a site-specific attack of guanosine on an oligonucleotide substrate. We previously determined that the hydrogen bond between the exocyclic amino group of guanosine and the 2'-hydroxyl group at position A261 of the Tetrahymena group I ribozyme contributes to overall catalysis. We now use functional data, aided by double mutant cycles, to probe this hydrogen bond in the individual reaction steps of the catalytic cycle. Our results indicate that this hydrogen bond is not formed upon guanosine binding to the ribozyme but instead forms at a later stage of the catalytic cycle. Formation of this hydrogen bond is correlated with other structural rearrangements in the ribozyme's active site that are promoted by docking of the oligonucleotide substrate into the ribozyme's active site, and disruption of this interaction has deleterious consequences for the chemical transformation within the ternary complex. These results, combined with earlier results, provide insight into the nature of the multiple conformational steps used by the Tetrahymena group I ribozyme to achieve its active structure and reveal an intricate, extended network of interactions that is used to establish catalytic interactions within this RNA's active site.

    View details for DOI 10.1021/bi902200n

    View details for Web of Science ID 000275858400015

    View details for PubMedID 20175542

  • Origins of catalysis by computationally designed retroaldolase enzymes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lassila, J. K., Baker, D., Herschlag, D. 2010; 107 (11): 4937-4942

    Abstract

    We have investigated recently reported computationally designed retroaldolase enzymes with the goal of understanding the extent and the origins of their catalytic power. Direct comparison of the designed enzymes to primary amine catalysts in solution revealed a rate acceleration of 10(5)-fold for the most active of the designed retroaldolases. Through pH-rate studies of the designed retroaldolases and evaluation of a Brønsted correlation for a series of amine catalysts, we found that lysine pK(a) values are shifted by 3-4 units in the enzymes but that the catalytic contributions from the shifted pK(a) values are estimated to be modest, about 10-fold. For the most active of the reported enzymes, we evaluated the catalytic contribution of two other design components: a motif intended to stabilize a bound water molecule and hydrophobic substrate binding interactions. Mutational analysis suggested that the bound water motif does not contribute to the rate acceleration. Comparison of the rate acceleration of the designed substrate relative to a minimal substrate suggested that hydrophobic substrate binding interactions contribute around 10(3)-fold to the enzymatic rate acceleration. Altogether, these results suggest that substrate binding interactions and shifting the pK(a) of the catalytic lysine can account for much of the enzyme's rate acceleration. Additional observations suggest that these interactions are limited in the specificity of placement of substrate and active site catalytic groups. Thus, future design efforts may benefit from a focus on achieving precision in binding interactions and placement of catalytic groups.

    View details for DOI 10.1073/pnas.0913638107

    View details for Web of Science ID 000275714300027

    View details for PubMedID 20194782

  • The Ligand-Free State of the TPP Riboswitch: A Partially Folded RNA Structure JOURNAL OF MOLECULAR BIOLOGY Ali, M., Lipfert, J., Seifert, S., Herschlag, D., Doniach, S. 2010; 396 (1): 153-165

    Abstract

    Riboswitches are elements of mRNA that regulate gene expression by undergoing structural changes upon binding of small ligands. Although the structures of several riboswitches have been solved with their ligands bound, the ligand-free states of only a few riboswitches have been characterized. The ligand-free state is as important for the functionality of the riboswitch as the ligand-bound form, but the ligand-free state is often a partially folded structure of the RNA, with conformational heterogeneity that makes it particularly challenging to study. Here, we present models of the ligand-free state of a thiamine pyrophosphate riboswitch that are derived from a combination of complementary experimental and computational modeling approaches. We obtain a global picture of the molecule using small-angle X-ray scattering data and use an RNA structure modeling software, MC-Sym, to fit local structural details to these data on an atomic scale. We have used two different approaches to obtaining these models. Our first approach develops a model of the RNA from the structures of its constituent junction fragments in isolation. The second approach treats the RNA as a single entity, without bias from the structure of its individual constituents. We find that both approaches give similar models for the ligand-free form, but the ligand-bound models differ for the two approaches, and only the models from the second approach agree with the ligand-bound structure known previously from X-ray crystallography. Our models provide a picture of the conformational changes that may occur in the riboswitch upon binding of its ligand. Our results also demonstrate the power of combining experimental small-angle X-ray scattering data with theoretical structure prediction tools in the determination of RNA structures beyond riboswitches.

    View details for DOI 10.1016/j.jmb.2009.11.030

    View details for Web of Science ID 000274766500013

    View details for PubMedID 19925806

  • Multiple native states reveal persistent ruggedness of an RNA folding landscape NATURE Solomatin, S. V., Greenfeld, M., Chu, S., Herschlag, D. 2010; 463 (7281): 681-U117

    Abstract

    According to the 'thermodynamic hypothesis', the sequence of a biological macromolecule defines its folded, active (or 'native') structure as a global energy minimum in the folding landscape. However, the enormous complexity of folding landscapes of large macromolecules raises the question of whether there is in fact a unique global minimum corresponding to a unique native conformation or whether there are deep local minima corresponding to alternative active conformations. The folding of many proteins is well described by two-state models, leading to highly simplified representations of protein folding landscapes with a single native conformation. Nevertheless, accumulating experimental evidence suggests a more complex topology of folding landscapes with multiple active conformations that can take seconds or longer to interconvert. Here we demonstrate, using single-molecule experiments, that an RNA enzyme folds into multiple distinct native states that interconvert on a timescale much longer than that of catalysis. These data demonstrate that severe ruggedness of RNA folding landscapes extends into conformational space occupied by native conformations.

    View details for DOI 10.1038/nature08717

    View details for Web of Science ID 000274193900040

    View details for PubMedID 20130651

  • Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kraut, D. A., Sigala, P. A., Fenn, T. D., Herschlag, D. 2010; 107 (5): 1960-1965

    Abstract

    The catalytic importance of enzyme active-site interactions is frequently assessed by mutating specific residues and measuring the resulting rate reductions. This approach has been used in bacterial ketosteroid isomerase to probe the energetic importance of active-site hydrogen bonds donated to the dienolate reaction intermediate. The conservative Tyr16Phe mutation impairs catalysis by 10(5)-fold, far larger than the effects of hydrogen bond mutations in other enzymes. However, the less-conservative Tyr16Ser mutation, which also perturbs the Tyr16 hydrogen bond, results in a less-severe 10(2)-fold rate reduction. To understand the paradoxical effects of these mutations and clarify the energetic importance of the Tyr16 hydrogen bond, we have determined the 1.6-A resolution x-ray structure of the intermediate analogue, equilenin, bound to the Tyr16Ser mutant and measured the rate effects of mutating Tyr16 to Ser, Thr, Ala, and Gly. The nearly identical 200-fold rate reductions of these mutations, together with the 6.4-A distance observed between the Ser16 hydroxyl and equilenin oxygens in the x-ray structure, strongly suggest that the more moderate rate effect of this mutant is not due to maintenance of a hydrogen bond from Ser at position 16. These results, additional spectroscopic observations, and prior structural studies suggest that the Tyr16Phe mutation results in unfavorable interactions with the dienolate intermediate beyond loss of a hydrogen bond, thereby exaggerating the apparent energetic benefit of the Tyr16 hydrogen bond relative to the solution reaction. These results underscore the complex energetics of hydrogen bonding interactions and site-directed mutagenesis experiments.

    View details for DOI 10.1073/pnas.0911168107

    View details for Web of Science ID 000274296300030

    View details for PubMedID 20080683

  • MEASURING THE ENERGETIC COUPLING OF TERTIARY CONTACTS IN RNA FOLDING USING SINGLE MOLECULE FLUORESCENCE RESONANCE ENERGY TRANSFER METHODS IN ENZYMOLOGY, VOL 472: SINGLE MOLECULE TOOLS, PT A: FLUORESCENCE BASED APPROACHES Greenfeld, M., Herschlag, D. 2010; 472: 205-220

    Abstract

    Tertiary contacts are critical to stabilizing the folded conformations of structured RNAs. In some cases, these contacts have been shown to interact with positive cooperativity. Measuring the energetic coupling of tertiary contact formation is among the most basic physical characterizations of a structured RNA. With proper experimental design, single-molecule fluorescence resonance energy transfer (smFRET) allows the rigorous determination of the energetic coupling. This chapter aims to provide a general experimental approach to measuring the energetic coupling of tertiary contacts, using smFRET.

    View details for DOI 10.1016/S0076-6879(10)72009-7

    View details for Web of Science ID 000279058600011

    View details for PubMedID 20580966

  • Do conformational biases of simple helical junctions influence RNA folding stability and specificity? RNA-A PUBLICATION OF THE RNA SOCIETY Chu, V. B., Lipfert, J., Bai, Y., Pande, V. S., Doniach, S., Herschlag, D. 2009; 15 (12): 2195-2205

    Abstract

    Structured RNAs must fold into their native structures and discriminate against a large number of alternative ones, an especially difficult task given the limited information content of RNA's nucleotide alphabet. The simplest motifs within structured RNAs are two helices joined by nonhelical junctions. To uncover the fundamental behavior of these motifs and to elucidate the underlying physical forces and challenges faced by structured RNAs, we computationally and experimentally studied a tethered duplex model system composed of two helices joined by flexible single- or double-stranded polyethylene glycol tethers, whose lengths correspond to those typically observed in junctions from structured RNAs. To dissect the thermodynamic properties of these simple motifs, we computationally probed how junction topology, electrostatics, and tertiary contact location influenced folding stability. Small-angle X-ray scattering was used to assess our predictions. Single- or double-stranded junctions, independent of sequence, greatly reduce the space of allowed helical conformations and influencing the preferred location and orientation of their adjoining helices. A double-stranded junction guides the helices along a hinge-like pathway. In contrast, a single-stranded junction samples a broader set of conformations and has different preferences than the double-stranded junction. In turn, these preferences determine the stability and distinct specificities of tertiary structure formation. These sequence-independent effects suggest that properties as simple as a junction's topology can generally define the accessible conformational space, thereby stabilizing desired structures and assisting in discriminating against misfolded structures. Thus, junction topology provides a fundamental strategy for transcending the limitations imposed by the low information content of RNA primary sequence.

    View details for DOI 10.1261/rna.1747509

    View details for Web of Science ID 000272169000011

    View details for PubMedID 19850914

  • Concordant Regulation of Translation and mRNA Abundance for Hundreds of Targets of a Human microRNA PLOS BIOLOGY Hendrickson, D. G., Hogan, D. J., McCullough, H. L., Myers, J. W., Herschlag, D., Ferrell, J. E., Brown, P. O. 2009; 7 (11)

    Abstract

    MicroRNAs (miRNAs) regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for approximately 8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For approximately 600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes) and ribosome density (the average number of ribosomes bound per unit length of coding sequence) were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124-mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the start site and are not consistent with inhibition of polypeptide elongation, or nascent polypeptide degradation contributing significantly to miRNA-mediated regulation in proliferating HEK293T cells. The observation of concordant changes in mRNA abundance and translational rate for hundreds of miR-124 targets is consistent with a functional link between these two regulatory outcomes of miRNA targeting, and the well-documented interrelationship between translation and mRNA decay.

    View details for DOI 10.1371/journal.pbio.1000238

    View details for Web of Science ID 000272032000004

    View details for PubMedID 19901979

  • Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schwans, J. P., Kraut, D. A., Herschlag, D. 2009; 106 (34): 14271-14275

    Abstract

    A fundamental difference between enzymes and small chemical catalysts is the ability of enzymes to use binding interactions with nonreactive portions of substrates to accelerate chemical reactions. Remote binding interactions can localize substrates to the active site, position substrates relative to enzymatic functional groups and other substrates, trigger conformational changes, induce local destabilization, and modulate an active site environment by solvent exclusion. We investigated the role of remote substrate binding interactions in the reaction catalyzed by the enzyme ketosteroid isomerase (KSI), which catalyzes a double bond migration of steroid substrates through a dienolate intermediate that is stabilized in an oxyanion hole. Comparison of a single-ring and multiple-ring substrate allowed the catalytic contribution of binding interactions with the distal substrate rings to be determined. The value of k(cat)/K(M) for a single-ring substrate is reduced 27,000-fold relative to a multiple-ring steroid substrate, suggesting that remote binding interactions with the steroid substrate contribute substantially to the KSI reaction. Nevertheless, the reaction rates for KSI-bound single- and multiple-ring substrates (k(cat)) are within 2-fold. Further, oxyanion hole mutations have the same effect on reactions of the single- and multiple-ring substrates. These results suggest that remote binding interactions contribute >5 kcal/mol to catalysis by KSI but that local rather than remote interactions dictate the catalytic contributions from KSI's general base and oxyanion hole.

    View details for DOI 10.1073/pnas.0901032106

    View details for Web of Science ID 000269295100021

    View details for PubMedID 19706511

  • The far reaches of enzymology NATURE CHEMICAL BIOLOGY Zalatan, J. G., Herschlag, D. 2009; 5 (8): 516-520

    View details for DOI 10.1038/nchembio0809-516

    View details for Web of Science ID 000268209200002

    View details for PubMedID 19620986

  • Hydrogen Bond Coupling in the Ketosteroid Isomerase Active Site BIOCHEMISTRY Sigala, P. A., Caaveiro, J. M., Ringe, D., Petsko, G. A., Herschlag, D. 2009; 48 (29): 6932-6939

    Abstract

    Hydrogen bond networks are key elements of biological structure and function. Nevertheless, their structural properties are challenging to assess within complex macromolecules. Hydrogen-bonded protons are not observed in the vast majority of protein X-ray structures, and static crystallographic models provide limited information regarding the dynamical coupling within hydrogen bond networks. We have brought together 1.1-1.3 A resolution X-ray crystallography, (1)H NMR, site-directed mutagenesis, and deuterium isotope effects on the geometry and chemical shifts of hydrogen-bonded protons to probe the conformational coupling of hydrogen bonds donated by Y16 and D103 in the oxyanion hole of bacterial ketosteroid isomerase. Our results suggest a robust physical coupling of the equilibrium structures of these two hydrogen bonds such that a lengthening of one hydrogen bond by as little as 0.01 A results in a shortening of the neighbor by a similar magnitude. Furthermore, the structural rearrangements detected by NMR in response to mutations within the active site hydrogen bond network can be explained on the basis of the observed coupling. The results herein elucidate fundamental structural properties of hydrogen bonds within the idiosyncratic environment of an enzyme active site and provide a foundation for future experimental and computational explorations of the role of coupled motions within hydrogen bond networks.

    View details for DOI 10.1021/bi900713j

    View details for Web of Science ID 000268175600023

    View details for PubMedID 19469568

  • Probing the Dynamics of the P1 Helix within the Tetrahymena Group I Intron JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Shi, X., Mollova, E. T., Pljevaljcic, G., Millar, D. P., Herschlag, D. 2009; 131 (27): 9571-9578

    Abstract

    RNA conformational transformations are integral to RNA's biological functions. Further, structured RNA molecules exist as a series of dynamic intermediates in the course of folding or complexation with proteins. Thus, an understanding of RNA folding and function will require deep and incisive understanding of its dynamic behavior. However, existing tools to investigate RNA dynamics are limited. Here, we introduce a powerful fluorescence polarization anisotropy approach that utilizes a rare base analogue that retains substantial fluorescence when incorporated into helices. We show that 6-methylisoxanthopterin (6-MI) can be used to follow the nanosecond dynamics of individual helices. We then use 6-MI to probe the dynamics of an individual helix, referred to as P1, within the 400nt Tetrahymena group I ribozyme. Comparisons of the dynamics of the P1 helix in wild type and mutant ribozymes and in model constructs reveal a highly immobilized docked state of the P1 helix, as expected, and a relatively mobile "open complex" or undocked state. This latter result rules out a model in which slow docking of the P1 helix into its cognate tertiary interactions arises from a stable alternatively docked conformer. The results are consistent with a model in which stacking and tertiary interactions of the A(3) tether connecting the P1 helix to the body of the ribozyme limit P1 mobility and slow its docking, and this model is supported by cross-linking results. The ability to isolate the nanosecond motions of individual helices within complex RNAs and RNA/protein complexes will be valuable in distinguishing between functional models and in discerning the fundamental behavior of important biological species.

    View details for DOI 10.1021/ja902797j

    View details for Web of Science ID 000268239400026

    View details for PubMedID 19537712

  • Hydrogen bond dynamics in the active site of photoactive yellow protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sigala, P. A., Tsuchida, M. A., Herschlag, D. 2009; 106 (23): 9232-9237

    Abstract

    Hydrogen bonds play major roles in biological structure and function. Nonetheless, hydrogen-bonded protons are not typically observed by X-ray crystallography, and most structural studies provide limited insight into the conformational plasticity of individual hydrogen bonds or the dynamical coupling present within hydrogen bond networks. We report the NMR detection of the hydrogen-bonded protons donated by Tyr-42 and Glu-46 to the chromophore oxygen in the active site of the bacterial photoreceptor, photoactive yellow protein (PYP). We have used the NMR resonances for these hydrogen bonds to probe their conformational properties and ability to rearrange in response to nearby electronic perturbation. The detection of geometric isotope effects transmitted between the Tyr-42 and Glu-46 hydrogen bonds provides strong evidence for robust coupling of their equilibrium conformations. Incorporation of a modified chromophore containing an electron-withdrawing cyano group to delocalize negative charge from the chromophore oxygen, analogous to the electronic rearrangement detected upon photon absorption, results in a lengthening of the Tyr-42 and Glu-46 hydrogen bonds and an attenuated hydrogen bond coupling. The results herein elucidate fundamental properties of hydrogen bonds within the complex environment of a protein interior. Furthermore, the robust conformational coupling and plasticity of hydrogen bonds observed in the PYP active site may facilitate the larger-scale dynamical coupling and signal transduction inherent to the biological function that PYP has evolved to carry out and may provide a model for other coupled dynamic systems.

    View details for DOI 10.1073/pnas.0900168106

    View details for Web of Science ID 000266817500025

    View details for PubMedID 19470452

  • Evaluating the Potential for Halogen Bonding in the Oxyanion Hole of Ketosteroid Isomerase Using Unnatural Amino Acid Mutagenesis ACS CHEMICAL BIOLOGY Kraut, D. A., Churchill, M. J., Dawson, P. E., Herschlag, D. 2009; 4 (4): 269-273

    Abstract

    There has recently been an increasing interest in controlling macromolecular conformations and interactions through halogen bonding. Halogen bonds are favorable electrostatic interactions between polarized, electropositive chlorine, bromine, or iodine atoms and electronegative atoms such as oxygen or nitrogen. These interactions have been likened to hydrogen bonds in terms of their favored acceptor molecules, their geometries, and their energetics. We asked whether a halogen bond could replace a hydrogen bond in the oxyanion hole of ketosteroid isomerase, using semisynthetic enzymes containing para-halogenated phenylalanine derivatives to replace the tyrosine hydrogen bond donor. Formation of a halogen bond to the oxyanion in the transition state would be expected to rescue the effects of mutation to phenylalanine, but all of the halogenated enzymes were comparable in activity to the phenylalanine mutant. We conclude that, at least in this active site, a halogen bond cannot functionally replace a hydrogen bond.

    View details for DOI 10.1021/cb900016q

    View details for Web of Science ID 000265444200006

    View details for PubMedID 19260691

  • Motions of the Substrate Recognition Duplex in a Group I Intron Assessed by Site-Directed Spin Labeling JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Grant, G. P., Boyd, N., Herschlag, D., Qin, P. Z. 2009; 131 (9): 3136-?

    Abstract

    The Tetrahymena group I intron recognizes its oligonucleotide substrate in a two-step process. First, a substrate recognition duplex, called the P1 duplex, is formed. The P1 duplex then docks into the prefolded ribozyme core by forming tertiary contacts. P1 docking controls both the rate and the fidelity of substrate cleavage and has been extensively studied as a model for the formation of RNA tertiary structure. However, previous work has been limited to studying millisecond or slower motions. Here we investigated nanosecond P1 motions in the context of the ribozyme using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A nitroxide spin label (R5a) was covalently attached to a specific site of the substrate oligonucleotide, the labeled substrate was bound to a prefolded ribozyme to form the P1 duplex, and X-band EPR spectroscopy was used to monitor nitroxide motions in the 0.1-50 ns regime. Using substrates that favor the docked or the undocked states, it was established that R5a was capable of reporting P1 duplex motions. Using R5a-labeled substrates it was found that the J1/2 junction connecting P1 to the ribozyme core controls nanosecond P1 mobility in the undocked state. This may account for previous observations that J1/2 mutations weaken substrate binding and give rise to cryptic cleavage. This study establishes the use of SDSL to probe nanosecond dynamic behaviors of individual helices within large RNA and RNA/protein complexes. This approach may help in understanding the relationship between RNA structure, dynamics, and function.

    View details for DOI 10.1021/ja808217s

    View details for Web of Science ID 000264792400008

    View details for PubMedID 19220053

  • Coarse-grained modeling of large RNA molecules with knowledge-based potentials and structural filters RNA-A PUBLICATION OF THE RNA SOCIETY Jonikas, M. A., Radmer, R. J., Laederach, A., Das, R., Pearlman, S., Herschlag, D., Altman, R. B. 2009; 15 (2): 189-199

    Abstract

    Understanding the function of complex RNA molecules depends critically on understanding their structure. However, creating three-dimensional (3D) structural models of RNA remains a significant challenge. We present a protocol (the nucleic acid simulation tool [NAST]) for RNA modeling that uses an RNA-specific knowledge-based potential in a coarse-grained molecular dynamics engine to generate plausible 3D structures. We demonstrate NAST's capabilities by using only secondary structure and tertiary contact predictions to generate, cluster, and rank structures. Representative structures in the best ranking clusters averaged 8.0 +/- 0.3 A and 16.3 +/- 1.0 A RMSD for the yeast phenylalanine tRNA and the P4-P6 domain of the Tetrahymena thermophila group I intron, respectively. The coarse-grained resolution allows us to model large molecules such as the 158-residue P4-P6 or the 388-residue T. thermophila group I intron. One advantage of NAST is the ability to rank clusters of structurally similar decoys based on their compatibility with experimental data. We successfully used ideal small-angle X-ray scattering data and both ideal and experimental solvent accessibility data to select the best cluster of structures for both tRNA and P4-P6. Finally, we used NAST to build in missing loops in the crystal structures of the Azoarcus and Twort ribozymes, and to incorporate crystallographic data into the Michel-Westhof model of the T. thermophila group I intron, creating an integrated model of the entire molecule. Our software package is freely available at https://simtk.org/home/nast.

    View details for DOI 10.1261/rna.1270809

    View details for Web of Science ID 000262463200001

    View details for PubMedID 19144906

  • Biophysical, chemical, and functional probes of RNA structure, interactions and folding: Part A. Preface. Methods in enzymology Herschlag, D. 2009; 468: xv-?

    View details for DOI 10.1016/S0076-6879(09)68020-4

    View details for PubMedID 19925921

  • RNA. Preface. Methods in enzymology Herschlag, D. 2009; 469: xvii-?

    View details for DOI 10.1016/S0076-6879(09)69028-5

    View details for PubMedID 19925922

  • PROBING NUCLEIC ACID-ION INTERACTIONS WITH BUFFER EXCHANGE-ATOMIC EMISSION SPECTROSCOPY METHODS IN ENZYMOLOGY, VOL 469: BIOPHYSICAL, CHEMICAL, AND FUNCTIONAL PROBES OF RNA STRUCTURE, INTERACTIONS AND FOLDING, PT B Greenfeld, M., Herschlag, D. 2009; 469: 375-389

    Abstract

    The ion atmosphere of nucleic acids directly affects measured biochemical and biophysical properties. However, study of the ion atmosphere is difficult due to its diffuse and dynamic nature. Standard techniques available have significant limitations in sensitivity, specificity, and directness of the assays. Buffer exchange-atomic emission spectroscopy (BE-AES) was developed to overcome many of the limitations of previously available techniques. This technique can provide a complete accounting of all ions constituting the ionic atmosphere of a nucleic acid at thermodynamic equilibrium. Although initially developed for the study of the ion atmosphere of nucleic acids, BE-AES has also been applied to study site-bound ions in RNA and protein.

    View details for DOI 10.1016/S0076-6879(09)69018-2

    View details for Web of Science ID 000272797000018

    View details for PubMedID 20946799

  • Structure and Function Converge To Identify a Hydrogen Bond in a Group I Ribozyme Active Site ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Forconi, M., Sengupta, R. N., Liu, M., Sartorelli, A. C., Piccirilli, J. A., Herschlag, D. 2009; 48 (39): 7171-7175

    View details for DOI 10.1002/anie.200903006

    View details for Web of Science ID 000269979100006

    View details for PubMedID 19708048

  • USE OF PHOSPHOROTHIOATES TO IDENTIFY SITES OF METAL-ION BINDING IN RNA METHODS IN ENZYMOLOGY, VOL 468: BIOPHYSICAL, CHEMICAL, AND FUNCTIONAL PROBES OF RNA STRUCTURE, INTERACTIONS AND FOLDING, PT A Forconi, M., Herschlag, D. 2009; 468: 311-333

    Abstract

    Single atom substitutions provide an exceptional opportunity to investigate RNA structure and function. Replacing a phosphoryl oxygen with a sulfur represents one of the most common and powerful single atom substitutions and can be used to determine the sites of metal-ion binding. Using functional assays of ribozyme catalysis, based on pre-steady-state kinetics, it is possible to extend this analysis to the transition state, capturing ligands for catalytic metal ions in this fleeting state. In conjunction with data determined from X-ray crystallography, this technique can provide a picture of the environment surrounding catalytic metal ions in both the ground state and the transition state at atomic resolution. Here, we describe the principles of such analysis, explain limitations of the method, and provide a practical example based on our results with the Tetrahymena group I ribozyme.

    View details for DOI 10.1016/S0076-6879(09)68015-0

    View details for Web of Science ID 000272435500016

    View details for PubMedID 20946776

  • METAL ION-BASED RNA CLEAVAGE AS A STRUCTURAL PROBE METHODS IN ENZYMOLOGY, VOL 468: BIOPHYSICAL, CHEMICAL, AND FUNCTIONAL PROBES OF RNA STRUCTURE, INTERACTIONS AND FOLDING, PT A Forconi, M., Herschlag, D. 2009; 468: 91-106

    Abstract

    It is well established that many metal ions accelerate the spontaneous degradation of RNA. This property has been exploited in several ways to garner information about RNA structure, especially in regards to the location of site-specifically bound metal ions, the presence of defined structural motifs, and the occurrence of conformational changes in structured RNAs. In this chapter, we review this information, briefly giving strengths and limitations for each of these approaches. Finally, we provide a general protocol to perform metal ion-mediated cleavage of RNA.

    View details for DOI 10.1016/S0076-6879(09)68005-8

    View details for Web of Science ID 000272435500006

    View details for PubMedID 20946766

  • Riboswitch conformations revealed by small-angle X-ray scattering. Methods in molecular biology (Clifton, N.J.) Lipfert, J., Herschlag, D., Doniach, S. 2009; 540: 141-159

    Abstract

    Riboswitches are functional RNA molecules that control gene expression through conformational changes in response to small-molecule ligand binding. In addition, riboswitch 3D structure, like that of other RNA molecules, is dependent on cation-RNA interactions as the RNA backbone is highly negatively charged. Here, we show how small-angle X-ray scattering (SAXS) can be used to probe RNA conformations as a function of ligand and ion concentration. In a recent study of a glycine-binding tandem aptamer from Vibrio cholerae, we have used SAXS data and thermodynamic modeling to investigate how Mg(2+)-dependent folding and glycine binding are energetically coupled. In addition, we have employed ab initio shape reconstruction algorithms to obtain low-resolution models of the riboswitch structure from SAXS data under different solution conditions.

    View details for DOI 10.1007/978-1-59745-558-9_11

    View details for PubMedID 19381558

  • FLUORESCENCE POLARIZATION ANISOTROPY TO MEASURE RNA DYNAMICS METHODS IN ENZYMOLOGY, VOL 469: BIOPHYSICAL, CHEMICAL, AND FUNCTIONAL PROBES OF RNA STRUCTURE, INTERACTIONS AND FOLDING, PT B Shi, X., Herschlag, D. 2009; 469: 287-302

    Abstract

    RNA requires helical motion to fold and carry out its function. As RNA helical motion occurs on the nanosecond timescale, the timescale probed by fluorescence dyes, fluorescence polarization anisotropy (FPA) is a simple, yet powerful, technique to study helical dynamics in RNA. With the recent development of several fluorescent base analogs that have a nanosecond timescale lifetime in a duplex, FPA has begun to be used for characterizing RNA dynamics. Using the probe 6-methylisoxanthopterin (6-MI) as an example, we describe the procedure for carrying out FPA experiments on model oligonucleotide systems and in a complex RNA, the Tetrahymena group I intron. For smaller RNA systems, isolating the motion of the target helix from the overall tumbling of the whole RNA system is necessary, and nucleic acids binding proteins can be incorporated into the RNA system to increase the overall size of the system, slow the overall tumbling, and thereby reduce the anisotropy contribution from the overall tumbling to negligible. The procedure for incorporating one such protein, the Lac Repressor, is given as an example.

    View details for DOI 10.1016/S0076-6879(09)69014-5

    View details for Web of Science ID 000272797000014

    View details for PubMedID 20946795

  • METHODS OF SITE-SPECIFIC LABELING OF RNA WITH FLUORESCENT DYES METHODS IN ENZYMOLOGY, VOL 469: BIOPHYSICAL, CHEMICAL, AND FUNCTIONAL PROBES OF RNA STRUCTURE, INTERACTIONS AND FOLDING, PT B Solomatin, S., Herschlag, D. 2009; 469: 47-68

    Abstract

    Single molecule fluorescence techniques offer unique insights into mechanisms of conformational changes of RNA. Knowing how to make fluorescently labeled RNA molecules and understanding potential limitations of different labeling strategies is essential for successful implementation of single molecule fluorescence techniques. This chapter offers a step by step overview of the process of obtaining RNA constructs ready for single molecule measurements. Several alternative methods are described for each step, and ways of troubleshooting the most common problems, in particular, splinted RNA ligation, are suggested.

    View details for DOI 10.1016/S0076-6879(09)69003-0

    View details for Web of Science ID 000272797000003

    View details for PubMedID 20946784

  • Comparative Enzymology in the Alkaline Phosphatase Superfamily to Determine the Catalytic Role of an Active-Site Metal Ion JOURNAL OF MOLECULAR BIOLOGY Zalatan, J. G., Fenn, T. D., Herschlag, D. 2008; 384 (5): 1174-1189

    Abstract

    Mechanistic models for biochemical systems are frequently proposed from structural data. Site-directed mutagenesis can be used to test the importance of proposed functional sites, but these data do not necessarily indicate how these sites contribute to function. In this study, we applied an alternative approach to the catalytic mechanism of alkaline phosphatase (AP), a widely studied prototypical bimetallo enzyme. A third metal ion site in AP has been suggested to provide general base catalysis, but comparison of AP with an evolutionarily related enzyme casts doubt on this model. Removal of this metal site from AP has large differential effects on reactions of cognate and promiscuous substrates, and the results are inconsistent with general base catalysis. Instead, these and additional results suggest that the third metal ion stabilizes the transferred phosphoryl group in the transition state. These results establish a new mechanistic model for this prototypical bimetallo enzyme and demonstrate the power of a comparative approach for probing biochemical function.

    View details for DOI 10.1016/j.jmb.2008.09.059

    View details for Web of Science ID 000262016600014

    View details for PubMedID 18851975

  • Promiscuous Sulfatase Activity and Thio-Effects in a Phosphodiesterase of the Alkaline Phosphatase Superfamily BIOCHEMISTRY Lassila, J. K., Herschlag, D. 2008; 47 (48): 12853-12859

    Abstract

    The nucleotide phosphodiesterase/pyrophosphatase from Xanthomonas axonopodis (NPP) is a structural and evolutionary relative of alkaline phosphatase that preferentially hydrolyzes phosphate diesters. With the goal of understanding how these two enzymes with nearly identical Zn(2+) bimetallo sites achieve high selectivity for hydrolysis of either phosphate monoesters or diesters, we have measured a promiscuous sulfatase activity in NPP. Sulfate esters are nearly isosteric with phosphate esters but carry less charge, offering a probe of electrostatic contributions to selectivity. NPP exhibits sulfatase activity with k(cat)/K(M) value of 2 x 10(-5) M(-1) s(-1), similar to the R166S mutant of alkaline phosphatase. We further report the effects of thio-substitution on phosphate monoester and diester reactions. Reactivities with these noncognate substrates illustrate a reduced dependence of NPP reactivity on the charge of the nonbridging oxygen situated between the Zn(2+) ions relative to that in alkaline phosphatase. This reduced charge dependence can explain about 10(2) of the 10(7)-fold differential catalytic proficiency for the most similar monoester and diester substrates in the two enzymes. The results further suggest that active site contacts to substrate oxygen atoms that do not contact the Zn(2+) ions may play an important role in defining the selectivity of the enzymes.

    View details for DOI 10.1021/bi801488c

    View details for Web of Science ID 000261202300024

    View details for PubMedID 18975918

  • A repulsive field: advances in the electrostatics of the ion atmosphere CURRENT OPINION IN CHEMICAL BIOLOGY Chu, V. B., Bai, Y., Lipfert, J., Herschlag, D., Doniach, S. 2008; 12 (6): 619-625

    Abstract

    The large electrostatic repulsion arising from the negatively charged backbone of RNA molecules presents a large barrier to folding. Solution counterions assist in the folding process by screening this electrostatic repulsion. While early research interpreted the effect of these counterions in terms of an empirical ligand-binding model, theories based on physical models have supplanted them and revised our view of the roles that ions play in folding. Instead of specific ion-binding sites, most ions in solution interact inside an 'ion atmosphere'--a fluctuating cloud of nonspecifically associated ions surrounding any charged molecule. Recent advances in experiments have begun the task of characterizing the ion atmosphere, yielding valuable data that have revealed deficiencies in Poisson-Boltzmann theory, the most widely used theory of the ion atmosphere. The continued development of experiments will help guide the development of improved theories, with the ultimate goal of understanding RNA folding and function and nucleic acid/protein interactions from a quantitative perspective.

    View details for DOI 10.1016/j.cbpa.2008.10.010

    View details for Web of Science ID 000262541300004

    View details for PubMedID 19081286

  • Testing geometrical discrimination within an enzyme active site: Constrained hydrogen bonding in the ketosterold isomerase oxyanion hole JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sigala, P. A., Kraut, D. A., Caaveiro, J. M., Pybus, B., Ruben, E. A., Ringe, D., Petsko, G. A., Herschlag, D. 2008; 130 (41): 13696-13708

    Abstract

    Enzymes are classically proposed to accelerate reactions by binding substrates within active-site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1-1 A scale of most substrate rearrangements. The flexibility of active-site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together 1.2-1.5 A resolution X-ray crystallography, (1)H and (19)F NMR spectroscopy, quantum mechanical calculations, and transition-state analogue binding measurements to test the distance scale on which noncovalent forces can constrain the structural relaxation or translation of side chains and ligands along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side-chain reorientation and prevent hydrogen bond shortening by 0.1 A or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguishable in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function.

    View details for DOI 10.1021/ja803928m

    View details for Web of Science ID 000259924000040

    View details for PubMedID 18808119

  • Diverse RNA-Binding Proteins Interact with Functionally Related Sets of RNAs, Suggesting an Extensive Regulatory System PLOS BIOLOGY Hogan, D. J., Riordan, D. P., Gerber, A. P., Herschlag, D., Brown, P. O. 2008; 6 (10): 2297-2313

    Abstract

    RNA-binding proteins (RBPs) have roles in the regulation of many post-transcriptional steps in gene expression, but relatively few RBPs have been systematically studied. We searched for the RNA targets of 40 proteins in the yeast Saccharomyces cerevisiae: a selective sample of the approximately 600 annotated and predicted RBPs, as well as several proteins not annotated as RBPs. At least 33 of these 40 proteins, including three of the four proteins that were not previously known or predicted to be RBPs, were reproducibly associated with specific sets of a few to several hundred RNAs. Remarkably, many of the RBPs we studied bound mRNAs whose protein products share identifiable functional or cytotopic features. We identified specific sequences or predicted structures significantly enriched in target mRNAs of 16 RBPs. These potential RNA-recognition elements were diverse in sequence, structure, and location: some were found predominantly in 3'-untranslated regions, others in 5'-untranslated regions, some in coding sequences, and many in two or more of these features. Although this study only examined a small fraction of the universe of yeast RBPs, 70% of the mRNA transcriptome had significant associations with at least one of these RBPs, and on average, each distinct yeast mRNA interacted with three of the RBPs, suggesting the potential for a rich, multidimensional network of regulation. These results strongly suggest that combinatorial binding of RBPs to specific recognition elements in mRNAs is a pervasive mechanism for multi-dimensional regulation of their post-transcriptional fate.

    View details for DOI 10.1371/journal.pbio.0060255

    View details for Web of Science ID 000260423900021

    View details for PubMedID 18959479

  • Critical assessment of nucleic acid electrostatics via experimental and computational investigation of an unfolded state ensemble JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bai, Y., Chu, V. B., Lipfert, J., Pande, V. S., Herschlag, D., Doniach, S. 2008; 130 (37): 12334-12341

    Abstract

    Electrostatic forces, acting between helices and modulated by the presence of the ion atmosphere, are key determinants in the energetic balance that governs RNA folding. Previous studies have employed Poisson-Boltzmann (PB) theory to compute the energetic contribution of these forces in RNA folding. However, the complex interaction of these electrostatic forces with RNA features such as tertiary contact formation, specific ion-binding, and complex interhelical junctions present in prior studies precluded a rigorous evaluation of PB theory, especially in physiologically important Mg(2+) solutions. To critically assess PB theory, we developed a model system that isolates these electrostatic forces. The model system, composed of two DNA duplexes tethered by a polyethylene glycol junction, is an analog for the unfolded state of canonical helix-junction-helix motifs found in virtually all structured RNAs. This model system lacks the complicating features that have precluded a critical assessment of PB in prior studies, ensuring that interhelical electrostatic forces dominate the behavior of the system. The system's simplicity allows PB predictions to be directly compared with small-angle X-ray scattering experiments over a range of monovalent and divalent ion concentrations. These comparisons indicate that PB is a reasonable description of the underlying electrostatic energies for monovalent ions, but large deviations are observed for divalent ions. The validation of PB for monovalent solutions allows analysis of the change in the conformational ensemble of this simple motif as salt concentration is changed. Addition of ions allows the motif to sample more compact microstates, increasing its conformational entropy. The increase of conformational entropy presents an additional barrier to folding by stabilizing the unfolded state. Neglecting this effect will adversely impact the accuracy of folding analyses and models.

    View details for DOI 10.1021/ja800854u

    View details for Web of Science ID 000259139900046

    View details for PubMedID 18722445

  • Arginine coordination in enzymatic phosphoryl transfer: Evaluation of the effect of Arg166 mutations in Escherichia coli alkaline phosphatase BIOCHEMISTRY O'Brien, P. J., Lassila, J. K., Fenn, T. D., Zalatan, J. G., Herschlag, D. 2008; 47 (29): 7663-7672

    Abstract

    Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia coli alkaline phosphatase on hydrolysis of ethyl phosphate, on individual reaction steps in the hydrolysis of the covalent enzyme-phosphoryl intermediate, and on thio substitution effects. The results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, we construct a free energy profile for the hydrolysis of the enzyme-phosphate intermediate. This analysis indicates that the arginine side chain strengthens binding by approximately 3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state. A 2.1 A X-ray diffraction structure of Arg166Ser AP is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate. Altogether, these results support a model in which the arginine contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state.

    View details for DOI 10.1021/bi800545n

    View details for Web of Science ID 000257665100008

    View details for PubMedID 18627128

  • Functional identification of ligands for a catalytic metal ion in group I introns BIOCHEMISTRY Forconi, M., Lee, J., Lee, J. K., Piccirilli, J. A., Herschlag, D. 2008; 47 (26): 6883-6894

    Abstract

    Many enzymes use metal ions within their active sites to achieve enormous rate acceleration. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). The three-dimensional arrangement determined by X-ray crystallography provides a powerful starting point for identifying ground state interactions, but only functional studies can establish and interrogate transition state interactions. The Tetrahymena group I ribozyme is a paradigm for the study of RNA catalysis, and previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified catalytic metal ions making five contacts with the substrate atoms. Here, we have combined atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to establish transition state ligands on the ribozyme for one of the catalytic metal ions, referred to as M A. We identified the pro-S P oxygen atoms at nucleotides C208, A304, and A306 as ground state ligands for M A, verifying interactions suggested by the Azoarcus crystal structures. We further established that these interactions are present in the chemical transition state, a conclusion that requires functional studies, such as those carried out herein. Elucidating these active site connections is a crucial step toward an in-depth understanding of how specific structural features of the group I intron lead to catalysis.

    View details for DOI 10.1021/bi800519a

    View details for Web of Science ID 000257095400017

    View details for PubMedID 18517225

  • Coupling between ATP binding and DNA cleavage by DNA topoisomerase II - A unifying kinetic and structural mechanism JOURNAL OF BIOLOGICAL CHEMISTRY Mueller-Planitz, F., Herschlag, D. 2008; 283 (25): 17463-17476

    Abstract

    DNA topoisomerase II is a molecular machine that couples ATP hydrolysis to the transport of one DNA segment through a transient break in another segment. To learn about the energetic connectivity that underlies this coupling, we investigated how the ATPase domains exert control over DNA cleavage. We dissected the DNA cleavage reaction by measuring rate and equilibrium constants for the individual reaction steps utilizing defined DNA duplexes in the presence and absence of the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMPPNP). Our results revealed the existence of two enzyme conformations whose relative abundance is sensitive to the presence of nucleotides. The predominant species in the absence of nucleotides binds DNA at a diffusion limited rate but cannot efficiently cleave DNA. In the presence of AMPPNP, most of the enzyme is converted to a state in which DNA binding and release is extremely slow but which allows DNA cleavage. A minimal kinetic and thermodynamic framework is established that accounts for the cooperativity of cleavage of the two DNA strands in the presence and absence of bound AMPPNP and includes conformational steps revealed in the kinetic studies. The model unifies available kinetic, thermodynamic, and structural data to provide a description for the reaction in terms of the order and rate of individual reaction steps and the physical nature of the species on the reaction path. Furthermore, this reaction framework provides a foundation for a future in-depth analysis of energy transduction by topoisomerase II, for guiding and interpreting future structural studies, and for analyzing the mechanism of drugs that convert topoisomerase into a cellular poison.

    View details for DOI 10.1074/jbc.M710014200

    View details for Web of Science ID 000256720600054

    View details for PubMedID 18403371

  • Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs CURRENT OPINION IN STRUCTURAL BIOLOGY Chu, V. B., Herschlag, D. 2008; 18 (3): 305-314

    Abstract

    The rapid development of our understanding of the diverse biological roles fulfilled by non-coding RNA has motivated interest in the basic macromolecular behavior, structure, and function of RNA. We focus on two areas in the behavior of complex RNAs. First, we present advances in the understanding of how RNA folding is accomplished in vivo by presenting a mechanism for the action of DEAD-box proteins. Members of this family are intimately associated with almost all cellular processes involving RNA, mediating RNA structural rearrangements and chaperoning their folding. Next, we focus on advances in understanding, and characterizing the basic biophysical forces that govern the folding of complex RNAs. Ultimately we expect that a confluence and synergy between these approaches will lead to profound understanding of RNA and its biology.

    View details for DOI 10.1016/j.sbi.2008.05.002

    View details for Web of Science ID 000257539100005

    View details for PubMedID 18555681

  • Direct measurement of tertiary contact cooperativity in RNA folding JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sattint, B. D., Zhao, W., Travers, K., Chut, S., Herschlag, D. 2008; 130 (19): 6085-?

    Abstract

    All structured biological macromolecules must overcome the thermodynamic folding problem to populate a unique functional state among a vast ensemble of unfolded and alternate conformations. The exploration of cooperativity in protein folding has helped reveal and distinguish the underlying mechanistic solutions to this folding problem. Analogous dissections of RNA tertiary stability remain elusive, however, despite the central biological importance of folded RNA molecules and the potential to reveal fundamental properties of structured macromolecules via comparisons of protein and RNA folding. We report a direct quantitative measure of tertiary contact cooperativity in a folded RNA. We precisely measured the stability of an independently folding P4-P6 domain from the Tetrahymena thermophila group I intron by single molecule fluorescence resonance energy transfer (smFRET). Using wild-type and mutant RNAs, we found that cooperativity between the two tertiary contacts enhances P4-P6 stability by 3.2 +/- 0.2 kcal/mol.

    View details for DOI 10.1021/ja800919q

    View details for Web of Science ID 000255620200014

    View details for PubMedID 18429611

  • Systematic Identification of mRNAs Recruited to Argonaute 2 by Specific microRNAs and Corresponding Changes in Transcript Abundance PLOS ONE Hendrickson, D. G., Hogan, D. J., Herschlag, D., Ferrell, J. E., Brown, P. O. 2008; 3 (5)

    Abstract

    microRNAs (miRNAs) are small non-coding RNAs that regulate mRNA stability and translation through the action of the RNAi-induced silencing complex (RISC). Our current understanding of miRNA function is inferred largely from studies of the effects of miRNAs on steady-state mRNA levels and from seed match conservation and context in putative targets. Here we have taken a more direct approach to these issues by comprehensively assessing the miRNAs and mRNAs that are physically associated with Argonaute 2 (Ago2), which is a core RISC component. We transfected HEK293T cells with epitope-tagged Ago2, immunopurified Ago2 together with any associated miRNAs and mRNAs, and quantitatively determined the levels of these RNAs by microarray analyses. We found that Ago2 immunopurified samples contained a representative repertoire of the cell's miRNAs and a select subset of the cell's total mRNAs. Transfection of the miRNAs miR-1 and miR-124 caused significant changes in the association of scores of mRNAs with Ago2. The mRNAs whose association with Ago2 increased upon miRNA expression were much more likely to contain specific miRNA seed matches and to have their overall mRNA levels decrease in response to the miRNA transfection than expected by chance. Hundreds of mRNAs were recruited to Ago2 by each miRNA via seed sequences in 3'-untranslated regions and coding sequences and a few mRNAs appear to be targeted via seed sequences in 5'-untranslated regions. Microarray analysis of Ago2 immunopurified samples provides a simple, direct method for experimentally identifying the targets of miRNAs and for elucidating roles of miRNAs in cellular regulation.

    View details for DOI 10.1371/journal.pone.0002126

    View details for Web of Science ID 000261642400047

    View details for PubMedID 18461144

  • Structural inference of native and partially folded RNA by high-throughput contact mapping PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dast, R., Kudaravalli, M., Jonikas, M., Laederach, A., Fong, R., Schwans, J. P., Baker, D., Piccirilli, J. A., Altman, R. B., Herschlag, D. 2008; 105 (11): 4144-4149

    Abstract

    The biological behaviors of ribozymes, riboswitches, and numerous other functional RNA molecules are critically dependent on their tertiary folding and their ability to sample multiple functional states. The conformational heterogeneity and partially folded nature of most of these states has rendered their characterization by high-resolution structural approaches difficult or even intractable. Here we introduce a method to rapidly infer the tertiary helical arrangements of large RNA molecules in their native and non-native solution states. Multiplexed hydroxyl radical (.OH) cleavage analysis (MOHCA) enables the high-throughput detection of numerous pairs of contacting residues via random incorporation of radical cleavage agents followed by two-dimensional gel electrophoresis. We validated this technology by recapitulating the unfolded and native states of a well studied model RNA, the P4-P6 domain of the Tetrahymena ribozyme, at subhelical resolution. We then applied MOHCA to a recently discovered third state of the P4-P6 RNA that is stabilized by high concentrations of monovalent salt and whose partial order precludes conventional techniques for structure determination. The three-dimensional portrait of a compact, non-native RNA state reveals a well ordered subset of native tertiary contacts, in contrast to the dynamic but otherwise similar molten globule states of proteins. With its applicability to nearly any solution state, we expect MOHCA to be a powerful tool for illuminating the many functional structures of large RNA molecules and RNA/protein complexes.

    View details for DOI 10.1073/pnas.0709032105

    View details for Web of Science ID 000254263300015

    View details for PubMedID 18322008

  • Semiautomated and rapid quantification of nucleic acid footprinting and structure mapping experiments NATURE PROTOCOLS Laederach, A., Das, R., Vicens, Q., Pearlman, S. M., Brenowitz, M., Herschlag, D., Altman, R. B. 2008; 3 (9): 1395-1401

    Abstract

    We have developed protocols for rapidly quantifying the band intensities from nucleic acid chemical mapping gels at single-nucleotide resolution. These protocols are implemented in the software SAFA (semi-automated footprinting analysis) that can be downloaded without charge from http://safa.stanford.edu. The protocols implemented in SAFA have five steps: (i) lane identification, (ii) gel rectification, (iii) band assignment, (iv) model fitting and (v) band-intensity normalization. SAFA enables the rapid quantitation of gel images containing thousands of discrete bands, thereby eliminating a bottleneck to the analysis of chemical mapping experiments. An experienced user of the software can quantify a gel image in approximately 20 min. Although SAFA was developed to analyze hydroxyl radical (*OH) footprints, it effectively quantifies the gel images obtained with other types of chemical mapping probes. We also present a series of tutorial movies that illustrate the best practices and different steps in the SAFA analysis as a supplement to this protocol.

    View details for DOI 10.1038/nprot.2008.134

    View details for Web of Science ID 000258424100003

    View details for PubMedID 18772866

  • Quantitative and comprehensive decomposition of the ion atmosphere around nucleic acids JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bai, Y., Greenfeld, M., Travers, K. J., Chu, V. B., Lipfert, J., Doniach, S., Herschlag, D. 2007; 129 (48): 14981-14988

    Abstract

    The ion atmosphere around nucleic acids critically affects biological and physical processes such as chromosome packing, RNA folding, and molecular recognition. However, the dynamic nature of the ion atmosphere renders it difficult to characterize. The basic thermodynamic description of this atmosphere, a full accounting of the type and number of associated ions, has remained elusive. Here we provide the first complete accounting of the ion atmosphere, using buffer equilibration and atomic emission spectroscopy (BE-AES) to accurately quantitate the cation association and anion depletion. We have examined the influence of ion size and charge on ion occupancy around simple, well-defined DNA molecules. The relative affinity of monovalent and divalent cations correlates inversely with their size. Divalent cations associate preferentially over monovalent cations; e.g., with Na+ in 4-fold excess of Mg2+ (20 vs 5 mM), the ion atmosphere nevertheless has 3-fold more Mg2+ than Na+. Further, the dicationic polyamine putrescine2+ does not compete effectively for association relative to divalent metal ions, presumably because of its lower charge density. These and other BE-AES results can be used to evaluate and guide the improvement of electrostatic treatments. As a first step, we compare the BE-AES results to predictions from the widely used nonlinear Poisson Boltzmann (NLPB) theory and assess the applicability and precision of this theory. In the future, BE-AES in conjunction with improved theoretical models, can be applied to complex binding and folding equilibria of nucleic acids and their complexes, to parse the electrostatic contribution from the overall thermodynamics of important biological processes.

    View details for DOI 10.1021/ja075020g

    View details for Web of Science ID 000251293500034

    View details for PubMedID 17990882

  • Evaluation of ion binding to DNA duplexes using a size-modified Poisson-Boltzmann theory BIOPHYSICAL JOURNAL Chu, V. B., Bai, Y., Lipfert, J., Herschlag, D., Doniach, S. 2007; 93 (9): 3202-3209

    Abstract

    Poisson-Boltzmann (PB) theory is among the most widely applied electrostatic theories in biological and chemical science. Despite its reasonable success in explaining a wide variety of phenomena, it fails to incorporate two basic physical effects, ion size and ion-ion correlations, into its theoretical treatment. Recent experimental work has shown significant deviations from PB theory in competitive monovalent and divalent ion binding to a DNA duplex. The experimental data for monovalent binding are consistent with a hypothesis that attributes these deviations to counterion size. To model the observed differences, we have generalized an existing size-modified Poisson-Boltzmann (SMPB) theory and developed a new numerical implementation that solves the generalized theory around complex, atomistic representations of biological molecules. The results of our analysis show that good agreement to data at monovalent ion concentrations up to approximately 150 mM can be attained by adjusting the ion-size parameters in the new size-modified theory. SMPB calculations employing calibrated ion-size parameters predict experimental observations for other nucleic acid structures and salt conditions, demonstrating that the theory is predictive. We are, however, unable to model the observed deviations in the divalent competition data with a theory that only accounts for size but neglects ion-ion correlations, highlighting the need for theoretical descriptions that further incorporate ion-ion correlations. The accompanying numerical solver has been released publicly, providing the general scientific community the ability to compute SMPB solutions around a variety of different biological structures with only modest computational resources.

    View details for DOI 10.1529/biophysj.106.099168

    View details for Web of Science ID 000250199300024

    View details for PubMedID 17604318

  • Do ligand binding and solvent exclusion alter the electrostatic character within the oxyanion hole of an enzymatic active site? JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sigala, P. A., Fafarman, A. T., Bogard, P. E., Boxer, S. G., Herschlag, D. 2007; 129 (40): 12104-?

    View details for DOI 10.1021/ja075605a

    View details for Web of Science ID 000249949600025

    View details for PubMedID 17854190

  • Modulation of individual steps in group I intron catalysis by a peripheral metal ion RNA-A PUBLICATION OF THE RNA SOCIETY Forconi, M., Piccirilli, J. A., Herschlag, D. 2007; 13 (10): 1656-1667

    Abstract

    Enzymes are complex macromolecules that catalyze chemical reactions at their active sites. Important information about catalytic interactions is commonly gathered by perturbation or mutation of active site residues that directly contact substrates. However, active sites are engaged in intricate networks of interactions within the overall structure of the macromolecule, and there is a growing body of evidence about the importance of peripheral interactions in the precise structural organization of the active site. Here, we use functional studies, in conjunction with published structural information, to determine the effect of perturbation of a peripheral metal ion binding site on catalysis in a well-characterized catalytic RNA, the Tetrahymena thermophila group I ribozyme. We perturbed the metal ion binding site by site-specifically introducing a phosphorothioate substitution in the ribozyme's backbone, replacing the native ligands (the pro-R (P) oxygen atoms at positions 307 and 308) with sulfur atoms. Our data reveal that these perturbations affect several reaction steps, including the chemical step, despite the absence of direct contacts of this metal ion with the atoms involved in the chemical transformation. As structural probing with hydroxyl radicals did not reveal significant change in the three-dimensional structure upon phosphorothioate substitution, the effects are likely transmitted through local, rather subtle conformational rearrangements. Addition of Cd(2+), a thiophilic metal ion, rescues some reaction steps but has deleterious effects on other steps. These results suggest that native interactions in the active site may have been aligned by the naturally occurring peripheral residues and interactions to optimize the overall catalytic cycle.

    View details for DOI 10.1261/rna.632007

    View details for Web of Science ID 000249920600007

    View details for PubMedID 17720880

  • Kinetic isotope effects for alkaline phosphatase reactions: Implications for the role of active-site metal ions in catalysis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Zalatan, J. G., Catrina, I., Mitchell, R., Grzyska, P. K., O'Brien, P. J., Herschlag, D., Hengge, A. C. 2007; 129 (31): 9789-9798

    Abstract

    Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active-site functional groups. In particular, the phosphate monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyzes phosphate monoester hydrolysis through a loose transition state, similar to that in solution. To gain further insight into the nature of the transition state and active-site interactions, we have determined kinetic isotope effects (KIEs) for AP-catalyzed hydrolysis reactions with several phosphate monoester substrates. The LFER and KIE data together provide a consistent picture for the nature of the transition state for AP-catalyzed phosphate monoester hydrolysis and support previous models suggesting that the enzymatic transition state is similar to that in solution. Moreover, the KIE data provides unique information regarding specific interactions between the transition state and the active-site Zn2+ ions. These results provide strong support for a model in which electrostatic interactions between the bimetallo Zn2+ site and a nonbridging phosphate ester oxygen atom make a significant contribution to the large rate enhancement observed for AP-catalyzed phosphate monoester hydrolysis.

    View details for DOI 10.1021/ja072196+

    View details for Web of Science ID 000248484400061

    View details for PubMedID 17630738

  • Low specificity of metal ion binding in the metal ion core of a folded RNA RNA-A PUBLICATION OF THE RNA SOCIETY Travers, K. J., Boyd, N., Herschlag, D. 2007; 13 (8): 1205-1213

    Abstract

    The structure and activity of nucleic acids depend on their interactions with metal ions. Fundamental to these interactions is the degree of specificity observed between the metal ions and nucleic acids, and a complete description of nucleic acid folding requires that we understand the nature of the interactions with metal ions, including specificity. The prior demonstration that high concentrations of monovalent cations prevent nonspecific association of divalent ions with nucleic acids provides a novel and powerful means to examine site-specific metal ion binding isolated from complicating effects of the ion atmosphere. Using these high monovalent cation solution conditions we have monitored the affinity of a series of divalent metal ions for two site-specific metal ion binding sites in the P4-P6 domain of the Tetrahymena group I intron ribozyme. The metal ion core of this highly structured RNA binds two divalent metal ions under these conditions. Despite multiple metal ion-RNA interactions observed in the X-ray crystallographic structure of P4-P6 RNA at the metal ion binding sites, these sites exhibit low specificity among Mn(2+), Mg(2+), Ca(2+), Ni(2+), and Zn(2+). Nevertheless, the largest divalent metal ions tested, Sr(2+) and Ba(2+), were excluded from binding, exhibiting affinities at least two orders of magnitude weaker than observed for the other metal ions. Thus, a picture emerges of two metal ion binding sites, each with a high tolerance for metal ions with different properties but also with limits to accommodation. These limits presumably arise from steric or electrostatic features of the metal ion binding sites.

    View details for DOI 10.1261/rna.566007

    View details for Web of Science ID 000248093500008

    View details for PubMedID 17616553

  • DNA topoisomerase II selects DNA cleavage sites based on reactivity rather than binding affinity NUCLEIC ACIDS RESEARCH Mueller-Planitz, F., Herschlag, D. 2007; 35 (11): 3764-3773

    Abstract

    DNA topoisomerase II modulates DNA topology by relieving supercoil stress and by unknotting or decatenating entangled DNA. During its reaction cycle, the enzyme creates a transient double-strand break in one DNA segment, the G-DNA. This break serves as a gate through which another DNA segment is transported. Defined topoisomerase II cleavage sites in genomic and plasmid DNA have been previously mapped. To dissect the G-DNA recognition mechanism, we studied the affinity and reactivity of a series of DNA duplexes of varied sequence under conditions that only allow G-DNA to bind. These DNA duplexes could be cleaved to varying extents ranging from undetectable (<0.5%) to 80%. The sequence that defines a cleavage site resides within the central 20 bp of the duplex. The DNA affinity does not correlate with the ability of the enzyme to cleave DNA, suggesting that the binding step does not contribute significantly to the selection mechanism. Kinetic experiments show that the selectivity interactions are formed before rather than subsequent to cleavage. Presumably the binding energy of the cognate interactions is used to promote a conformational change that brings the enzyme into a cleavage competent state. The ability to modulate the extent of DNA cleavage by varying the DNA sequence may be valuable for future structural and mechanistic studies that aim to determine topoisomerase structures with DNA bound in pre- and post-cleavage states and to understand the conformational changes associated with DNA binding and cleavage.

    View details for DOI 10.1093/nar/gkm335

    View details for Web of Science ID 000247817500024

    View details for PubMedID 17517767

  • Probing the origin of the compromised catalysis of E-coli Alkaline phosphatase in its promiscuous sulfatase reaction JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Catrina, I., O'Brien, P. J., Purcell, J., Nikolic-Hughes, I., Zalatan, J. G., Hengge, A. C., Herschlag, D. 2007; 129 (17): 5760-5765

    Abstract

    The catalytic promiscuity of E. coli alkaline phosphatase (AP) and many other enzymes provides a unique opportunity to dissect the origin of enzymatic rate enhancements via a comparative approach. Here, we use kinetic isotope effects (KIEs) to explore the origin of the 109-fold greater catalytic proficiency by AP for phosphate monoester hydrolysis relative to sulfate monoester hydrolysis. The primary 18O KIEs for the leaving group oxygen atoms in the AP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) and p-nitrophenylsulfate (pNPS) decrease relative to the values observed for nonenzymatic hydrolysis reactions. Prior linear free energy relationship results suggest that the transition states for AP-catalyzed reactions of phosphate and sulfate esters are "loose" and indistinguishable from that in solution, suggesting that the decreased primary KIEs do not reflect a change in the nature of the transition state but rather a strong interaction of the leaving group oxygen atom with an active site Zn2+ ion. Furthermore, the primary KIEs for the two reactions are identical within error, suggesting that the differential catalysis of these reactions cannot be attributed to differential stabilization of the leaving group. In contrast, AP perturbs the KIE for the nonbridging oxygen atoms in the reaction of pNPP but not pNPS, suggesting a differential interaction with the transferred group in the transition state. These and prior results are consistent with a strong electrostatic interaction between the active site bimetallo Zn2+ cluster and one of the nonbridging oxygen atoms on the transferred group. We suggest that the lower charge density of this oxygen atom on a transferred sulfuryl group accounts for a large fraction of the decreased stabilization of the transition state for its reaction relative to phosphoryl transfer.

    View details for DOI 10.1021/ja069111+

    View details for Web of Science ID 000245946400086

    View details for PubMedID 17411045

  • Measuring the folding transition time of single RNA molecules BIOPHYSICAL JOURNAL Lee, T., Lapidus, L. J., Zhao, W., Travers, K. J., Herschlag, D., Chu, S. 2007; 92 (9): 3275-3283

    Abstract

    We describe a new, time-apertured photon correlation method for resolving the transition time between two states of RNA in folding--i.e., the time of the transition between states rather than the time spent in each state. Single molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy are used to obtain these measurements. Individual RNA molecules are labeled with fluorophores such as Cy3 and Cy5. Those molecules are then immobilized on a surface and observed for many seconds during which time the molecules spontaneously switch between two conformational states with different levels of flourescence resonance energy transfer efficiency. Single photons are counted from each fluorophore and cross correlated in a small window around a transition. The average of over 1000 cross correlations can be fit to a polynomial, which can determine transition times as short as the average photon emission interval. We applied the method to the P4-P6 domain of the Tetrahymena group I self-splicing intron to yield the folding transition time of 240 micros. The unfolding time is found to be too short to measure with this method.

    View details for DOI 10.1529/biophysj.106.094623

    View details for Web of Science ID 000245544100034

    View details for PubMedID 17307831

  • Probing the role of a secondary structure element at the 5 '- and 3 '-splice sites in group I intron self-splicing: The Tetrahymena L-16 ScaI ribozyme reveals a new role of the G center dot U pair in self-splicing BIOCHEMISTRY Karbstein, K., Lee, J., Herschlag, D. 2007; 46 (16): 4861-4875

    Abstract

    Several ribozyme constructs have been used to dissect aspects of the group I self-splicing reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogues, catalyzes a reaction analogous to the first step of self-splicing, in which a 5'-splice site analogue (S) and guanosine (G) are converted into a 5'-exon analogue (P) and GA. This ribozyme preserves the active site but lacks a short 5'-terminal segment (called the IGS extension herein) that forms dynamic helices, called the P1 extension and P10 helix. The P1 extension forms at the 5'-splice site in the first step of self-splicing, and P10 forms at the 3'-splice site in the second step of self-splicing. To dissect the contributions from the IGS extension and the helices it forms, we have investigated the effects of each of these elements at each reaction step. These experiments were performed with the L-16 ScaI ribozyme, which retains the IGS extension, and with 5'- and 3'-splice site analogues that differ in their ability to form the helices. The presence of the IGS extension strengthens binding of P by 40-fold, even when no new base pairs are formed. This large effect was especially surprising, as binding of S is essentially unaffected for S analogues that do not form additional base pairs with the IGS extension. Analysis of a U.U pair immediately 3' to the cleavage site suggests that a previously identified deleterious effect from a dangling U residue on the L-21 ScaI ribozyme arises from a fortuitous active site interaction and has implications for RNA tertiary structure specificity. Comparisons of the affinities of 5'-splice site analogues that form only a subset of base pairs reveal that inclusion of the conserved G.U base pair at the cleavage site of group I introns destabilizes the P1 extension >100-fold relative to the stability of a helix with all Watson-Crick base pairs. Previous structural data with model duplexes and the recent intron structures suggest that this effect can be attributed to partial unstacking of the P1 extension at the G.U step. These results suggest a previously unrecognized role of the G.U wobble pair in self-splicing: breaking cooperativity in base pair formation between P1 and the P1 extensions. This effect may facilitate replacement of the P1 extension with P10 after the first chemical step of self-splicing and release of the ligated exons after the second step of self-splicing.

    View details for DOI 10.1021/bi062169g

    View details for Web of Science ID 000245735900018

    View details for PubMedID 17385892

  • Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae JOURNAL OF MOLECULAR BIOLOGY Lipfert, J., Das, R., Chu, V. B., Kudaravalli, M., Boyd, N., Herschlag, D., Doniach, S. 2007; 365 (5): 1393-1406

    Abstract

    Riboswitches are complex folded RNA domains found in noncoding regions of mRNA that regulate gene expression upon small molecule binding. Recently, Breaker and coworkers reported a tandem aptamer riboswitch (VCI-II) that binds glycine cooperatively. Here, we use hydroxyl radical footprinting and small-angle X-ray scattering (SAXS) to study the conformations of this tandem aptamer as a function of Mg(2+) and glycine concentration. We fit a simple three-state thermodynamic model that describes the energetic coupling between magnesium-induced folding and glycine binding. Furthermore, we characterize the structural conformations of each of the three states: In low salt with no magnesium present, the VCI-II construct has an extended overall conformation, presumably representing unfolded structures. Addition of millimolar concentrations of Mg(2+) in the absence of glycine leads to a significant compaction and partial folding as judged by hydroxyl radical protections. In the presence of millimolar Mg(2+) concentrations, the tandem aptamer binds glycine cooperatively. The glycine binding transition involves a further compaction, additional tertiary packing interactions and further uptake of magnesium ions relative to the state in high Mg(2+) but no glycine. Employing density reconstruction algorithms, we obtain low resolution 3-D structures for all three states from the SAXS measurements. These data provide a first glimpse into the structural conformations of the VCI-II aptamer, establish rigorous constraints for further modeling, and provide a framework for future mechanistic studies.

    View details for DOI 10.1016/j.jmb.2006.10.022

    View details for Web of Science ID 000243749600013

    View details for PubMedID 17118400

  • Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid SCIENCE Woodside, M. T., Anthony, P. C., Behnke-Parks, W. M., Larizadeh, K., Herschlag, D., Block, S. M. 2006; 314 (5801): 1001-1004

    Abstract

    Nucleic acid hairpins provide a powerful model system for understanding macromolecular folding, with free-energy landscapes that can be readily manipulated by changing the hairpin sequence. The full shapes of energy landscapes for the reversible folding of DNA hairpins under controlled loads exerted by an optical force clamp were obtained by deconvolution from high-resolution, single-molecule trajectories. The locations and heights of the energy barriers for hairpin folding could be tuned by adjusting the number and location of G:C base pairs, and the presence and position of folding intermediates were controlled by introducing single-nucleotide mismatches.

    View details for DOI 10.1126/science.1133601

    View details for Web of Science ID 000241896000056

    View details for PubMedID 17095702

  • The paradoxical behavior of a highly structured misfolded intermediate in RNA folding JOURNAL OF MOLECULAR BIOLOGY Russell, R., Das, R., Suh, H., Traver, K. J., Laederach, A., Engelhardt, M. A., Herschlag, D. 2006; 363 (2): 531-544

    Abstract

    Like many structured RNAs, the Tetrahymena group I ribozyme is prone to misfolding. Here we probe a long-lived misfolded species, referred to as M, and uncover paradoxical aspects of its structure and folding. Previous work indicated that a non-native local secondary structure, termed alt P3, led to formation of M during folding in vitro. Surprisingly, hydroxyl radical footprinting, fluorescence measurements with site-specifically incorporated 2-aminopurine, and functional assays indicate that the native P3, not alt P3, is present in the M state. The paradoxical behavior of alt P3 presumably arises because alt P3 biases folding toward M, but, after commitment to this folding pathway and before formation of M, alt P3 is replaced by P3. Further, structural and functional probes demonstrate that the misfolded ribozyme contains extensive native structure, with only local differences between the two states, and the misfolded structure even possesses partial catalytic activity. Despite the similarity of these structures, re-folding of M to the native state is very slow and is strongly accelerated by urea, Na+, and increased temperature and strongly impeded by Mg2+ and the presence of native peripheral contacts. The paradoxical observations of extensive native structure within the misfolded species but slow conversion of this species to the native state are readily reconciled by a model in which the misfolded state is a topological isomer of the native state, and computational results support the feasibility of this model. We speculate that the complex topology of RNA secondary structures and the inherent rigidity of RNA helices render kinetic traps due to topological isomers considerably more common for RNA than for proteins.

    View details for DOI 10.1016/j.jmb.2006.08.024

    View details for Web of Science ID 000241324500019

    View details for PubMedID 16963081

  • The wide reach of enzymology: from bioorganic chemistry to chemical biology and beyond mechanisms - Editorial overview CURRENT OPINION IN CHEMICAL BIOLOGY Fierke, C. A., Herschlag, D. 2006; 10 (5): 453-454
  • Interdomain communication in DNA topoisomerase II - DNA binding and enzyme activation JOURNAL OF BIOLOGICAL CHEMISTRY Mueller-Planitz, F., Herschlag, D. 2006; 281 (33): 23395-23404

    Abstract

    Topoisomerase II catalyzes the ATP-dependent transport of a DNA segment (T-DNA) through a transient double strand break in another DNA segment (G-DNA). A fundamental mechanistic question is how the individual steps in this process are coordinated. We probed communication between the DNA binding sites and the individual enzymatic activities, ATP hydrolysis, and DNA cleavage. We employed short DNA duplexes to control occupancy at the two binding sites of wild-type enzyme and a variant with a G-DNA site mutation. The DNA concentration dependence of ATP hydrolysis and a fluorescence anisotropy assay provided thermodynamic information about DNA binding. The results suggest that G-DNA binds with higher affinity than T-DNA. Enzyme with only G-DNA bound is competent to cleave DNA, indicating that T-DNA is dispensable for DNA cleavage. The ATPase activity of enzyme bound solely to G-DNA is partially stimulated. Full stimulation requires binding of T-DNA. Both DNA binding sites therefore signal to the ATPase domains. The results support and extend current mechanistic models for topoisomerase II-catalyzed DNA transport and provide a framework for future mechanistic dissection.

    View details for DOI 10.1074/jbc.M604119200

    View details for Web of Science ID 000239702900014

    View details for PubMedID 16782968

  • Structural and functional comparisons of nucleotide pyrophosphatase/phosphodiesterase and alkaline phosphatase: Implications for mechanism and evolution BIOCHEMISTRY Zalatan, J. G., Fenn, T. D., Brunger, A. T., Herschlag, D. 2006; 45 (32): 9788-9803

    Abstract

    The rapid expansion of the amount of genomic and structural data has provided many examples of enzymes with evolutionarily related active sites that catalyze different reactions. Functional comparisons of these active sites can provide insight into the origins of the enormous catalytic proficiency of enzymes and the evolutionary changes that can lead to different enzyme activities. The alkaline phosphatase (AP) superfamily is an ideal system to use in making such comparisons given the extensive data available on both nonenzymatic and enzymatic phosphoryl transfer reactions. Some superfamily members, such as AP itself, preferentially hydrolyze phosphate monoesters, whereas others, such as nucleotide pyrophosphatase/phosphodiesterase (NPP), preferentially hydrolyze phosphate diesters. We have measured rate constants for NPP-catalyzed hydrolysis of phosphate diesters and monoesters. NPP preferentially catalyzes diester hydrolysis by factors of 10(2)-10(6), depending on the identity of the diester substrate. To identify features of the NPP active site that could lead to preferential phosphate diester hydrolysis, we have determined the structure of NPP in the absence of ligands and in complexes with vanadate and AMP. Comparisons to existing structures of AP reveal bimetallo cores that are structurally indistinguishable, but there are several distinct structural features outside of the conserved bimetallo site. The structural and functional data together suggest that some of these distinct functional groups provide specific substrate binding interactions, whereas others tune the properties of the bimetallo active site itself to discriminate between phosphate diester and monoester substrates.

    View details for DOI 10.1021/bi060847t

    View details for Web of Science ID 000239596600016

    View details for PubMedID 16893180

  • Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Woodside, M. T., Behnke-Parks, W. M., Larizadeh, K., Travers, K., Herschlag, D., Block, S. M. 2006; 103 (16): 6190-6195

    Abstract

    Nucleic acid hairpins provide a powerful model system for probing the formation of secondary structure. We report a systematic study of the kinetics and thermodynamics of the folding transition for individual DNA hairpins of varying stem length, loop length, and stem GC content. Folding was induced mechanically in a high-resolution optical trap using a unique force clamp arrangement with fast response times. We measured 20 different hairpin sequences with quasi-random stem sequences that were 6-30 bp long, polythymidine loops that were 3-30 nt long, and stem GC content that ranged from 0% to 100%. For all hairpins studied, folding and unfolding were characterized by a single transition. From the force dependence of these rates, we determined the position and height of the energy barrier, finding that the transition state for duplex formation involves the formation of 1-2 bp next to the loop. By measuring unfolding energies spanning one order of magnitude, transition rates covering six orders of magnitude, and hairpin opening distances with subnanometer precision, our results define the essential features of the energy landscape for folding. We find quantitative agreement over the entire range of measurements with a hybrid landscape model that combines thermodynamic nearest-neighbor free energies and nanomechanical DNA stretching energies.

    View details for DOI 10.1073/pnas.0511048103

    View details for Web of Science ID 000236999000025

    View details for PubMedID 16606839

  • Testing electrostatic complementarity in enzyme catalysis: Hydrogen bonding in the ketosteroid isomerase oxyanion hole PLOS BIOLOGY Kraut, D. A., Sigala, P. A., Pybus, B., Liu, C. W., Ringe, D., Petsko, G. A., Herschlag, D. 2006; 4 (4): 501-519

    Abstract

    A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing pK(a) models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50-0.76 ppm/pK(a) unit, suggesting a bond shortening of 0.02 A/pK(a) unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (DeltaDeltaG = -0.2 kcal/mol/pK(a) unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (DeltaDeltaH = -2.0 kcal/mol/pK(a) unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of 300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution.

    View details for DOI 10.1371/journal.pbio.0040099

    View details for Web of Science ID 000237066500005

    View details for PubMedID 16602823

  • Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gerber, A. P., Luschnig, S., Krasnow, M. A., Brown, P. O., Herschlag, D. 2006; 103 (12): 4487-4492

    Abstract

    Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. PUMILIO is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. We generated transgenic flies expressing affinity-tagged PUMILIO under the control of an ovary-specific promoter, and we purified PUMILIO from whole adult flies and embryos and analyzed associated mRNAs by using DNA microarrays. Distinct sets comprising hundreds of mRNAs were associated with PUMILIO at the two developmental stages. Many of these mRNAs encode functionally related proteins, supporting a model for coordinated regulation of posttranscriptional modules by specific RNA-binding proteins. We identified a characteristic sequence motif in the 3'-untranslated regions of mRNAs associated with PUMILIO, and the sufficiency of this motif for interaction with PUMILIO was confirmed by RNA pull-down experiments with biotinylated synthetic RNAs. The RNA motif strikingly resembles the one previously identified for Puf3p, one of five Saccharomyces cerevisiae Puf proteins; however, proteins encoded by the associated mRNAs in yeast and Drosophila do not appear to be related. The results suggest extensive posttranscriptional regulation by PUMILIO and uncover evolutionary features of this conserved family of RNA-binding proteins.

    View details for DOI 10.1073/pnas.0509260103

    View details for Web of Science ID 000236362600031

    View details for PubMedID 16537387

  • Alkaline phosphatase mono- and diesterase reactions: Comparative transition state analysis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Zalatan, J. G., Herschlag, D. 2006; 128 (4): 1293-1303

    Abstract

    Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts. Previous work with Escherichia coli alkaline phosphatase (AP), however, suggests that this enzyme catalyzes the hydrolysis of phosphate monoesters through a loose, dissociative transition state, similar to that in solution. AP also exhibits catalytic promiscuity, with a low level of phosphodiesterase activity, despite the tighter, more associative transition state for phosphate diester hydrolysis in solution. Because AP is evolutionarily optimized for phosphate monoester hydrolysis, it is possible that the active site environment alters the transition state for diester hydrolysis to be looser in its bonding to the incoming and outgoing groups. To test this possibility, we have measured the nonenzymatic and AP-catalyzed rate of reaction for a series of substituted methyl phenyl phosphate diesters. The values of beta(lg) and additional data suggest that the transition state for AP-catalyzed phosphate diester hydrolysis is indistinguishable from that in solution. Instead of altering transition state structure, AP catalyzes phosphoryl transfer reactions by recognizing and stabilizing transition states similar to those in aqueous solution. The AP active site therefore has the ability to recognize different transition states, a property that could assist in the evolutionary optimization of promiscuous activities.

    View details for DOI 10.1021/ja056528r

    View details for Web of Science ID 000234941100056

    View details for PubMedID 16433548

  • Functional identification of catalytic metal ion binding sites within RNA PLOS BIOLOGY Hougland, J. L., Kravchuk, A. V., Herschlag, D., Piccirilli, J. A. 2005; 3 (9): 1536-1548

    Abstract

    The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-SP phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.

    View details for DOI 10.1371/journal.pbio.0030277

    View details for Web of Science ID 000231820900005

    View details for PubMedID 16092891

  • Structural specificity conferred by a group I RNA peripheral element PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Johnson, T. H., Tijerina, P., Chadee, A. B., Herschlag, D., Russell, R. 2005; 102 (29): 10176-10181

    Abstract

    Like proteins, structured RNAs must specify a native conformation that is more stable than all other possible conformations. Local structure is much more stable for RNA than for protein, so it is likely that the principal challenge for RNA is to stabilize the native structure relative to misfolded and partially folded intermediates rather than unfolded structures. Many structured RNAs contain peripheral structural elements, which surround the core elements. Although it is clear that peripheral elements stabilize structure within RNAs that contain them, it has not yet been explored whether they specifically stabilize the native states relative to alternative folds. A two-piece version of the group I intron RNA from Tetrahymena is used here to show that the peripheral element P5abc binds to the native conformation of the rest of the RNA 50,000 times more tightly than it binds to a long-lived misfolded conformation. Thus, P5abc stabilizes the native conformation by approximately 6 kcal/mol relative to this misfolded conformation. Further, activity measurements show that for the RNA lacking P5abc, the native conformation is only marginally preferred over the misfolded conformation (<0.5 kcal/mol), indicating that the peripheral structure of this RNA is required to achieve a significant thermodynamic preference for the native state. Such "structural specificity" may be a general function of RNA peripheral domains.

    View details for DOI 10.1073/pnas.0501498102

    View details for Web of Science ID 000230665800035

    View details for PubMedID 16009943

  • Alkaline phosphatase catalysis is ultrasensitive to charge sequestered between the active site zinc ions JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Nikolic-Hughes, I., O'Brien, P. J., Herschlag, D. 2005; 127 (26): 9314-9315

    Abstract

    Escherichia coli alkaline phosphatase (AP) is a prototypical bimetalloenzyme, facilitating catalysis of phosphate monoester hydrolysis with two Zn2+ metal ions that are only 4 A apart. In the reaction's transition state, one of the nonbridging oxygen atoms of the transferred group appears to interact directly with the Zn2+ ion metallocluster. To determine the importance and the energetic properties of this interaction, we systematically varied the charge on this oxygen atom, exploiting the ability of AP to catalyze reactions of different classes of substrates. We observed that the AP catalytic proficiency correlates very well (R2 = 0.98) with the charge on this oxygen atom, over 8 orders of magnitude of catalytic proficiency. The slope of this linear correlation (31 +/- 2 kcal/mol per unit charge) is extraordinarily steep, indicating that AP greatly discriminates between differentially charged substrates. We suggest that this discrimination arises via an electrostatic interaction with the bimetallocluster. The dependence of the AP catalytic proficiency on the nonbridging oxygen charge is much larger than charge perturbation effects observed previously for other proteins. We propose that AP uses folding energy to position the two Zn2+ metal ions in close proximity, thereby creating an active site with a high electrostatic potential that is extraordinarily sensitive to the charge that "solvates" the metallocluster. The sensitivity of enzyme energetics to systematic variation in electrostatic properties provides a powerful measure of the active site environment. Future work comparing the sensitivity of related enzymes that have been optimized to catalyze different reactions will help reveal how natural selection has tuned related active sites to favor different reactions.

    View details for DOI 10.1021/ja051603j

    View details for Web of Science ID 000230220900002

    View details for PubMedID 15984827

  • Determining the Mg2+ stoichiometry for folding an RNA metal ion core JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Das, R., Travers, K. J., Bai, Y., Herschlag, D. 2005; 127 (23): 8272-8273

    Abstract

    The folding and catalytic function of RNA molecules depend on their interactions with divalent metal ions, such as magnesium. As with every molecular process, the most basic knowledge required for understanding the close relationship of an RNA with its metal ions is the stoichiometry of the interaction. Unfortunately, inventories of the numbers of divalent ions associated with unfolded and folded RNA states have been unattainable. A common approach has been to interpret Hill coefficients fit to folding equilibria as the number of metal ions bound upon folding. However, this approach is vitiated by the presence of diffusely associated divalent ions in a dynamic ion atmosphere and by the likelihood of multiple transitions along a folding pathway. We demonstrate that the use of molar concentrations of background monovalent salt can alleviate these complications. These simplifying solution conditions allow a precise determination of the stoichiometry of the magnesium ions involved in folding the metal ion core of the P4-P6 domain of the Tetrahymena group I ribozyme. Hill analysis of hydroxyl radical footprinting data suggests that the P4-P6 RNA core folds cooperatively upon the association of two metal ions. This unexpectedly small stoichiometry is strongly supported by counting magnesium ions associated with the P4-P6 RNA via fluorescence titration and atomic emission spectroscopy. By pinpointing the metal ion stoichiometry, these measurements provide a critical but previously missing step in the thermodynamic dissection of the coupling between metal ion binding and RNA folding.

    View details for DOI 10.1021/ja051422h

    View details for Web of Science ID 000229751100020

    View details for PubMedID 15941246

  • Promiscuous catalysis by the Tetrahymena group I ribozyme JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Forconi, M., Herschlag, D. 2005; 127 (17): 6160-6161

    Abstract

    Catalytic promiscuity, the ability of an enzyme to catalyze alternative reactions, has been suggested to have played an important role in the evolution of new catalytic activities in protein enzymes. Similarly, promiscuous activities may have been advantageous in an earlier RNA world. The Tetrahymena Group I ribozyme naturally catalyzes the site-specific guanosine attack on an anionic phosphate diester and has been shown to also catalyze aminoacyl transfer to water, albeit with a small rate acceleration (<10-fold). This inefficient catalysis could be due to the differences in charge and/or geometry requirements for the two reactions. Herein, we describe a new promiscuous activity of this ribozyme, the site-specific guanosine attack on a neutral phosphonate diester. This alternative substrate lacks the negative charge at the reaction center but, in contrast to the aminoacyl substrate, can undergo nucleophilic attack with the same geometry as the natural substrate. Our results show that the neutral phosphonate reaction is catalyzed about 1 x 106-fold, substantially better than the acyl transfer but far below the normal anionic substrate. We conclude that both charge and geometry are important factors for catalysis of the normal reaction and that promiscuous catalytic activities of ribozymes could have been created or enhanced by reorienting and swapping RNA domains.

    View details for Web of Science ID 000228755300012

    View details for PubMedID 15853307

  • SAFA: Semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments RNA-A PUBLICATION OF THE RNA SOCIETY Das, R., Laederach, A., Pearlman, S. M., Herschlag, D., Altman, R. B. 2005; 11 (3): 344-354

    Abstract

    Footprinting is a powerful and widely used tool for characterizing the structure, thermodynamics, and kinetics of nucleic acid folding and ligand binding reactions. However, quantitative analysis of the gel images produced by footprinting experiments is tedious and time-consuming, due to the absence of informatics tools specifically designed for footprinting analysis. We have developed SAFA, a semi-automated footprinting analysis software package that achieves accurate gel quantification while reducing the time to analyze a gel from several hours to 15 min or less. The increase in analysis speed is achieved through a graphical user interface that implements a novel methodology for lane and band assignment, called "gel rectification," and an optimized band deconvolution algorithm. The SAFA software yields results that are consistent with published methodologies and reduces the investigator-dependent variability compared to less automated methods. These software developments simplify the analysis procedure for a footprinting gel and can therefore facilitate the use of quantitative footprinting techniques in nucleic acid laboratories that otherwise might not have considered their use. Further, the increased throughput provided by SAFA may allow a more comprehensive understanding of molecular interactions. The software and documentation are freely available for download at http://safa.stanford.edu.

    View details for DOI 10.1261/rna.7214405

    View details for Web of Science ID 000227190000011

    View details for PubMedID 15701734

  • Probing counterion modulated repulsion and attraction between nucleic acid duplexes in solution PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bai, Y., Das, R., Millett, I. S., Herschlag, D., Doniach, S. 2005; 102 (4): 1035-1040

    Abstract

    Understanding biological and physical processes involving nucleic acids, such as the binding of proteins to DNA and RNA, DNA condensation, and RNA folding, requires an understanding of the ion atmosphere that surrounds nucleic acids. We have used a simple model DNA system to determine how the ion atmosphere modulates interactions between duplexes in the absence of specific metal ion-binding sites and other complicated interactions. In particular, we have tested whether the Coulomb repulsion between nucleic acids can be reversed by counterions to give a net attraction, as has been proposed recently for the rapid collapse observed early in RNA folding. The conformation of two DNA duplexes tethered by a flexible neutral linker was determined in the presence of a series of cations by small angle x-ray scattering. The small angle x-ray scattering profiles of two control molecules with distinct shapes (a continuous duplex and a mimic of the compact DNA) were in good agreement with predictions, establishing the applicability of this approach. Under low-salt conditions (20 mM Na+), the tethered duplexes are extended because of a Coulombic repulsion estimated to be 2-5 kT/bp. Addition of high concentrations of Na+ (1.2 M), Mg2+ (0.6 M), and spermidine3+ (75 mM) resulted in electrostatic relaxation to a random state. These results indicate that a counterion-induced attractive force between nucleic acid duplexes is not significant under physiological conditions. An upper limit on the magnitude of the attractive potential under all tested ionic conditions is estimated.

    View details for DOI 10.1073/pnas.0404448102

    View details for Web of Science ID 000226617900016

    View details for PubMedID 15647360

  • Dissecting eukaryotic translation and its control by ribosome density mapping NUCLEIC ACIDS RESEARCH Arava, Y., Boas, F. E., Brown, P. O., Herschlag, D. 2005; 33 (8): 2421-2432

    Abstract

    Translation of an mRNA is generally divided into three stages: initiation, elongation and termination. The relative rates of these steps determine both the number and position of ribosomes along the mRNA, but traditional velocity sedimentation assays for the translational status of mRNA determine only the number of bound ribosomes. We developed a procedure, termed Ribosome Density Mapping (RDM), that uses site-specific cleavage of polysomal mRNA followed by separation on a sucrose gradient and northern analysis, to determine the number of ribosomes associated with specified portions of a particular mRNA. This procedure allows us to test models for translation and its control, and to examine properties of individual steps of translation in vivo. We tested specific predictions from the current model for translational control of GCN4 expression in yeast and found that ribosomes were differentially associated with the uORFs elements and coding region under different growth conditions, consistent with this model. We also mapped ribosome density along the ORF of several mRNAs, to probe basic kinetic properties of translational steps in yeast. We found no detectable decline in ribosome density between the 5' and 3' ends of the ORFs, suggesting that the average processivity of elongation is very high. Conversely, there was no queue of ribosomes at the termination site, suggesting that termination is not very slow relative to elongation and initiation. Finally, the RDM results suggest that less frequent initiation of translation on mRNAs with longer ORFs is responsible for the inverse correlation between ORF length and ribosomal density that we observed in a global analysis of translation. These results provide new insights into eukaryotic translation in vivo.

    View details for DOI 10.1093/nar/gki331

    View details for Web of Science ID 000229113100017

    View details for PubMedID 15860778

  • Spatial distribution of competing ions around DNA in solution PHYSICAL REVIEW LETTERS Andresen, K., Das, R., Park, H. Y., Smith, H., Kwok, L. W., Lamb, J. S., Kirkland, E. J., Herschlag, D., Finkelstein, K. D., Pollack, L. 2004; 93 (24)

    Abstract

    The competition of monovalent and divalent cations for proximity to negatively charged DNA is of biological importance and can provide strong constraints for theoretical treatments of polyelectrolytes. Resonant x-ray scattering experiments have allowed us to monitor the number and distribution of each cation in a mixed ion cloud around DNA. These measurements provide experimental evidence to support a general theoretical prediction: the normalized distribution of each ion around polyelectrolytes remains constant when ions are mixed at different ratios. In addition, the amplitudes of the scattering signals throughout the competition provide a measurement of the surface concentration parameter that predicts the competition behavior of these cations. The data suggest that ion size needs to be taken into account in applying Poisson-Boltzmann treatments to polyelectrolytes such as DNA.

    View details for DOI 10.1103/PhysRevLett.93.248103

    View details for Web of Science ID 000225661100083

    View details for PubMedID 15697865

  • Principles of RNA compaction: Insights from the equilibrium folding pathway of the P4-P6 RNA domain in monovalent cations JOURNAL OF MOLECULAR BIOLOGY Takamoto, K., Das, R., He, Q., Doniach, S., Brenowitz, M., Herschlag, D., Chance, M. R. 2004; 343 (5): 1195-1206

    Abstract

    Counterions are required for RNA folding, and divalent metal ions such as Mg(2+) are often critical. To dissect the role of counterions, we have compared global and local folding of wild-type and mutant variants of P4-P6 RNA derived from the Tetrahymena group I ribozyme in monovalent and in divalent metal ions. A remarkably simple picture of the folding thermodynamics emerges. The equilibrium folding pathway in monovalent ions displays two phases. In the first phase, RNA molecules that are initially in an extended conformation enforced by charge-charge repulsion are relaxed by electrostatic screening to a state with increased flexibility but without formation of long-range tertiary contacts. At higher concentrations of monovalent ions, a state that is nearly identical to the native folded state in the presence of Mg(2+) is formed, with tertiary contacts that involve base and backbone interactions but without the subset of interactions that involve specific divalent metal ion-binding sites. The folding model derived from these and previous results provides a robust framework for understanding the equilibrium and kinetic folding of RNA.

    View details for DOI 10.1016/j.jmb.2004.08.080

    View details for Web of Science ID 000224838800004

    View details for PubMedID 15491606

  • A base triple in the Tetrahymena group I core affects the reaction equilibrium via a threshold effect RNA-A PUBLICATION OF THE RNA SOCIETY Karbstein, K., Tang, K. H., Herschlag, D. 2004; 10 (11): 1730-1739

    Abstract

    Previous work on group I introns has suggested that a central base triple might be more important for the first rather than the second step of self-splicing, leading to a model in which the base triple undergoes a conformational change during self-splicing. Here, we use the well-characterized L-21 ScaI ribozyme derived from the Tetrahymena group I intron to probe the effects of base-triple disruption on individual reaction steps. Consistent with previous results, reaction of a ternary complex mimicking the first chemical step in self-splicing is slowed by mutations in this base triple, whereas reaction of a ternary complex mimicking the second step of self-splicing is not. Paradoxically, mechanistic dissection of the base-triple disruption mutants indicates that active site binding is weakened uniformly for the 5'-splice site and the 5'-exon analog, mimics for the species bound in the first and second step of self-splicing. Nevertheless, the 5'-exon analog remains bound at the active site, whereas the 5'-splice site analog does not. This differential effect arises despite the uniform destabilization, because the wild-type ribozyme binds the 5'-exon analog more strongly in the active site than in the 5'-splice site analog. Thus, binding into the active site constitutes an additional barrier to reaction of the 5'-splice site analog, but not the 5'-exon analog, resulting in a reduced reaction rate constant for the first step analog, but not the second step analog. This threshold model explains the self-splicing observations without the need to invoke a conformational change involving the base triple, and underscores the importance of quantitative dissection for the interpretation of effects from mutations.

    View details for DOI 10.1261/rna.7118104

    View details for Web of Science ID 000224746300009

    View details for PubMedID 15496521

  • Do electrostatic interactions with positively charged active site groups tighten the transition state for enzymatic phosphoryl transfer? JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Nikolic-Hughes, I., Rees, D. C., Herschlag, D. 2004; 126 (38): 11814-11819

    Abstract

    The effect of electrostatic interactions on the transition-state character for enzymatic phosphoryl transfer has been a subject of much debate. In this work, we investigate the transition state for alkaline phosphatase (AP) using linear free-energy relationships (LFERs). We determined k(cat)/K(M) for a series of aryl sulfate ester monoanions to obtain the Brønsted coefficient, beta(lg), and compared the value to that obtained previously for a series of aryl phosphorothioate ester dianion substrates. Despite the difference in substrate charge, the observed Brønsted coefficients for AP-catalyzed aryl sulfate and aryl phosphorothioate hydrolysis (-0.76 +/- 0.14 and -0.77 +/- 0.10, respectively) are strikingly similar, with steric effects being responsible for the uncertainties in these values. Aryl sulfates and aryl phosphates react via similar loose transition states in solution. These observations suggest an apparent equivalency of the transition states for phosphorothioate and sulfate hydrolysis reactions at the AP active site and, thus, negligible effects of active site electrostatic interactions on charge distribution in the transition state.

    View details for DOI 10.1021/ja0480421

    View details for Web of Science ID 000224103900032

    View details for PubMedID 15382915

  • Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast PLOS BIOLOGY Gerber, A. P., Herschlag, D., Brown, P. O. 2004; 2 (3): 342-354
  • Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast. PLoS biology Gerber, A. P., Herschlag, D., Brown, P. O. 2004; 2 (3): E79-?

    Abstract

    Genes encoding RNA-binding proteins are diverse and abundant in eukaryotic genomes. Although some have been shown to have roles in post-transcriptional regulation of the expression of specific genes, few of these proteins have been studied systematically. We have used an affinity tag to isolate each of the five members of the Puf family of RNA-binding proteins in Saccharomyces cerevisiae and DNA microarrays to comprehensively identify the associated mRNAs. Distinct groups of 40-220 different mRNAs with striking common themes in the functions and subcellular localization of the proteins they encode are associated with each of the five Puf proteins: Puf3p binds nearly exclusively to cytoplasmic mRNAs that encode mitochondrial proteins; Puf1p and Puf2p interact preferentially with mRNAs encoding membrane-associated proteins; Puf4p preferentially binds mRNAs encoding nucleolar ribosomal RNA-processing factors; and Puf5p is associated with mRNAs encoding chromatin modifiers and components of the spindle pole body. We identified distinct sequence motifs in the 3'-untranslated regions of the mRNAs bound by Puf3p, Puf4p, and Puf5p. Three-hybrid assays confirmed the role of these motifs in specific RNA-protein interactions in vivo. The results suggest that combinatorial tagging of transcripts by specific RNA-binding proteins may be a general mechanism for coordinated control of the localization, translation, and decay of mRNAs and thus an integral part of the global gene expression program.

    View details for PubMedID 15024427

  • Adenosine 5 '-O-(3-thio)triphosphate (ATP-gamma S) is a substrate for the nucleotide hydrolysis and RNA unwinding activities of eukaryotic translation initiation factor eIF4A RNA-A PUBLICATION OF THE RNA SOCIETY Peck, M. L., Herschlag, D. 2003; 9 (10): 1180-1187

    Abstract

    Whereas ATPgammaS is often considered a nonhydrolyzable substrate for ATPases, we present evidence that ATPgammaS is a good substrate for the RNA-stimulated nucleotide hydrolysis and RNA unwinding activities of eIF4A. In the presence of saturating single-stranded poly(U) RNA, eIF4A hydrolyzes ATPgammaS.Mg and ATP.Mg with similar steady-state parameters (KM(NTP.Mg) = 66 and 58 microM and kcat = 1.0 and 0.97 min(-1), respectively). ATPgammaS.Mg also supports catalysis of RNA unwinding within 10-fold of the rate supported by ATP.Mg. The identical steady-state rate parameters, in comparison with the expected difference in the intrinsic rate of hydrolysis for ATP and ATPgammaS, suggest a nonchemical rate-limiting step for nucleotide hydrolysis. These results raise caution concerning the assumption that ATPgammaS is a nonhydrolyzable ATP analog and underscore the utility of thio-substituted NTPs as mechanistic probes.

    View details for DOI 10.1261/rna.2103703

    View details for Web of Science ID 000185471400003

    View details for PubMedID 13130132

  • Widespread cytoplasmic mRNA transport in yeast: Identification of 22 bud-localized transcripts using DNA microarray analysis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shepard, K. A., Gerber, A. P., Jambhekar, A., Takizawa, P. A., Brown, P. O., Herschlag, D., DeRisi, J. L., Vale, R. D. 2003; 100 (20): 11429-11434

    Abstract

    Cytoplasmic mRNA localization provides a means of generating cell asymmetry and segregating protein activity. Previous studies have identified two mRNAs that localize to the bud tips of the yeast Saccharomyces cerevisiae. To identify additional localized mRNAs, we immunoprecipitated the RNA transport components She2p, She3p, and Myo4p and performed DNA microarray analysis of their associated RNAs. A secondary screen, using a GFP-tagged RNA reporter assay, identified 22 mRNAs that are localized to bud tips. These messages encode a wide variety of proteins, including several involved in stress responses and cell wall maintenance. Many of these proteins are asymmetrically localized to buds. However, asymmetric localization also occurs in the absence of RNA transport, suggesting the existence of redundant protein localization mechanisms. In contrast to findings in metazoans, the untranslated regions are dispensable for mRNA localization in yeast. This study reveals an unanticipated widespread use of RNA transport in budding yeast.

    View details for DOI 10.1073/pnas.2033246100

    View details for Web of Science ID 000185685700047

    View details for PubMedID 13679573

  • The fastest global events in RNA folding: Electrostatic relaxation and tertiary collapse of the tetrahymena ribozyme JOURNAL OF MOLECULAR BIOLOGY Das, R., Kwok, L. W., Millett, I. S., Bai, Y., Mills, T. T., JACOB, J., Maskel, G. S., Seifert, S., Mochrie, S. G., Thiyagarajan, P., Doniach, S., Pollack, L., Herschlag, D. 2003; 332 (2): 311-319

    Abstract

    Large RNAs can collapse into compact conformations well before the stable formation of the tertiary contacts that define their final folds. This study identifies likely physical mechanisms driving these early compaction events in RNA folding. We have employed time-resolved small-angle X-ray scattering to monitor the fastest global shape changes of the Tetrahymena ribozyme under different ionic conditions and with RNA mutations that remove long-range tertiary contacts. A partial collapse in each of the folding time-courses occurs within tens of milliseconds with either monovalent or divalent cations. Combined with comparison to predictions from structural models, this observation suggests a relaxation of the RNA to a more compact but denatured conformational ensemble in response to enhanced electrostatic screening at higher ionic concentrations. Further, the results provide evidence against counterion-correlation-mediated attraction between RNA double helices, a recently proposed model for early collapse. A previous study revealed a second 100 ms phase of collapse to a globular state. Surprisingly, we find that progression to this second early folding intermediate requires RNA sequence motifs that eventually mediate native long-range tertiary interactions, even though these regions of the RNA were observed to be solvent-accessible in previous footprinting studies under similar conditions. These results help delineate an analogy between the early conformational changes in RNA folding and the "burst phase" changes and molten globule formation in protein folding.

    View details for DOI 10.1016/S0022-2836(03)00854-4

    View details for Web of Science ID 000185306700002

    View details for PubMedID 12948483

  • Exploration of the transition state for tertiary structure formation between an RNA helix and a large structured RNA JOURNAL OF MOLECULAR BIOLOGY Bartley, L. E., Zhuang, X. W., Das, R., Chu, S., Herschlag, D. 2003; 328 (5): 1011-1026

    Abstract

    Docking of the P1 duplex into the pre-folded core of the Tetrahymena group I ribozyme exemplifies the formation of tertiary interactions in the context of a complex, structured RNA. We have applied Phi-analysis to P1 docking, which compares the effects of modifications on the rate constant for docking (k(dock)) with the effects on the docking equilibrium (K(dock)). To accomplish this we used a single molecule fluorescence resonance energy transfer assay that allows direct determination of the rate constants for formation of thermodynamically favorable, as well as unfavorable, states. Modification of the eight groups of the P1 duplex that make tertiary interactions with the core and changes in solution conditions decrease K(dock) up to 500-fold, whereas k(dock) changes by

    View details for Web of Science ID 000182767000004

    View details for PubMedID 12729738

  • Counterion distribution around DNA probed by solution X-ray scattering PHYSICAL REVIEW LETTERS Das, R., Mills, T. T., Kwok, L. W., Maskel, G. S., Millett, I. S., Doniach, S., Finkelstein, K. D., Herschlag, D., Pollack, L. 2003; 90 (18)

    Abstract

    Counterion atmospheres condensed onto charged biopolymers strongly affect their physical properties and biological functions, but have been difficult to quantify experimentally. Here, monovalent and divalent counterion atmospheres around DNA double helices in solution are probed using small-angle x-ray scattering techniques. Modulation of the ion scattering factors by anomalous (resonant) x-ray scattering and by interchanging ion identities yields direct measurements of the scattering signal due to the spatial correlation of surrounding ions to the DNA. The quality of the data permit, for the first time, quantitative tests of extended counterion distributions calculated from atomic-scale models of biologically relevant molecules.

    View details for DOI 10.1103/PhysRevLett.90.188103

    View details for Web of Science ID 000182823900054

    View details for PubMedID 12786045

  • Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Arava, Y., Wang, Y. L., Storey, J. D., Liu, C. L., Brown, P. O., Herschlag, D. 2003; 100 (7): 3889-3894

    Abstract

    We have analyzed the translational status of each mRNA in rapidly growing Saccharomyces cerevisiae. mRNAs were separated by velocity sedimentation on a sucrose gradient, and 14 fractions across the gradient were analyzed by quantitative microarray analysis, providing a profile of ribosome association with mRNAs for thousands of genes. For most genes, the majority of mRNA molecules were associated with ribosomes and presumably engaged in translation. This systematic approach enabled us to recognize genes with unusual behavior. For 43 genes, most mRNA molecules were not associated with ribosomes, suggesting that they may be translationally controlled. For 53 genes, including GCN4, CPA1, and ICY2, three genes for which translational control is known to play a key role in regulation, most mRNA molecules were associated with a single ribosome. The number of ribosomes associated with mRNAs increased with increasing length of the putative protein-coding sequence, consistent with longer transit times for ribosomes translating longer coding sequences. The density at which ribosomes were distributed on each mRNA (i.e., the number of ribosomes per unit ORF length) was well below the maximum packing density for nearly all mRNAs, consistent with initiation as the rate-limiting step in translation. Global analysis revealed an unexpected correlation: Ribosome density decreases with increasing ORF length. Models to account for this surprising observation are discussed.

    View details for DOI 10.1073/pnas.0635171100

    View details for Web of Science ID 000182058400066

    View details for PubMedID 12660367

  • Extraordinarily slow binding of guanosine to the Tetrahymena group I ribozyme: Implications for RNA preorganization and function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Karbstein, K., Herschlag, D. 2003; 100 (5): 2300-2305

    Abstract

    The Tetrahymena ribozyme derived from the self-splicing group I intron binds a 5'-splice site analog (S) and guanosine (G), catalyzing their conversion to a 5'-exon analog (P) and GA. Herein, we show that binding of guanosine is exceptionally slow, limiting the reaction at near neutral pH. Our results implicate a conformational rearrangement on guanosine binding, likely because the binding site is not prearranged in the absence of ligand. The fast accommodation of guanosine (10(2) to 10(3) x s(-1)) and prior structural data suggest local rather than global rearrangements, raising the possibility that folding of this and perhaps other large RNAs is not fully cooperative. Guanosine binding is accelerated by addition of residues that form helices, referred to as P9.0 and P10, immediately 5' and 3' to the guanosine. These rate enhancements provide evidence for binding intermediates that have the adjacent helices formed before accommodation of guanosine into its binding site. Because the ability to form the P9.0 and P10 helices distinguishes the guanosine at the correct 3'-splice site from other guanosine residues, the faster binding of the correct guanosine can enhance specificity of 3'-splice site selection. Thus, paradoxically, the absence of a preformed binding site and the resulting slow guanosine binding can contribute to splicing specificity by providing an opportunity for the adjacent helices to increase the rate of binding of the guanosine specifying the 3'-splice site.

    View details for DOI 10.1073/pnas.252749799

    View details for Web of Science ID 000181365000027

    View details for PubMedID 12591943

  • Challenges in enzyme mechanism and energetics ANNUAL REVIEW OF BIOCHEMISTRY Kraut, D. A., Carroll, K. S., Herschlag, D. 2003; 72: 517-571

    Abstract

    Since the discovery of enzymes as biological catalysts, study of their enormous catalytic power and exquisite specificity has been central to biochemistry. Nevertheless, there is no universally accepted comprehensive description. Rather, numerous proposals have been presented over the past half century. The difficulty in developing a comprehensive description for the catalytic power of enzymes derives from the highly cooperative nature of their energetics, which renders impossible a simple division of mechanistic features and an absolute partitioning of catalytic contributions into independent and energetically additive components. Site-directed mutagenesis has emerged as an enormously powerful approach to probe enzymatic catalysis, illuminating many basic features of enzyme function and behavior. The emphasis of site-directed mutagenesis on the role of individual residues has also, inadvertently, limited experimental and conceptual attention to the fundamentally cooperative nature of enzyme function and energetics. The first part of this review highlights the structural and functional interconnectivity central to enzymatic catalysis. In the second part we ask: What are the features of enzymes that distinguish them from simple chemical catalysts? The answers are presented in conceptual models that, while simplified, help illustrate the vast amount known about how enzymes achieve catalysis. In the last section, we highlight the molecular and energetic questions that remain for future investigation and describe experimental approaches that will be necessary to answer these questions. The promise of advancing and integrating cutting edge conceptual, experimental, and computational tools brings mechanistic enzymology to a new era, one poised for novel fundamental insights into biological catalysis.

    View details for DOI 10.1146/annurev.biochem.72.121801.161617

    View details for Web of Science ID 000185092500017

    View details for PubMedID 12704087

  • Environmental effects on phosphoryl group bonding probed by vibrational spectroscopy: Implications for understanding phosphoryl transfer and enzymatic catalysis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Cheng, H., Nikolic-Hughes, I., Wang, J. H., Deng, H., O'Brien, P. J., Wu, L., Zhang, Z. Y., Herschlag, D., Callender, R. 2002; 124 (38): 11295-11306

    Abstract

    We have used vibrational spectroscopy to study bonding in monosubstituted dianionic phosphates, both to learn more about basic properties intrinsic to this important class of biological substrates and to assess the ability of vibrational spectroscopy to provide a "sensor" or probe of the local environment experienced by the phosphoryl group. We examined the bonding properties of the phosphoryl group via vibrational spectroscopy for a series of compounds in which the phosphoryl substituent was varied systematically and extensively. A broad linear correlation of the bridging P-O(R) bond length and the pK(a) of the substituent alcohol was observed. The results indicate that the P-O(R) bond changes by only approximately 0.04 A with alcohol substituents that vary in pK(a) by approximately 12 units, suggesting that phosphoryl group bonding responds in a subtle but regular manner to changes in the local environment. We also determined the effect on the phosphoryl bonding from changes in the solvent environment. Addition of dimethyl sulfoxide (DMSO) elongates the bridging bond, presumably as a result of lessened solvation to the nonbridging oxygens and conservation of bond order to phosphorus. Finally, we have addressed the relationship between ground-state bonding properties and reactivity, as changing the leaving group substituent and adding DMSO have large rate effects, and it was previously proposed that lengthening of the bond to be broken is the cause of the increased reactivity. The results herein suggest, however, that the change in the bridging bond energy is small compared to the changes in energy that accompany the observed reactivity differences. Further analysis indicates that electrostatic interactions can provide a common driving force underlying both bond lengthening and the observed rate increases. We suggest that ground-state distortions of substrates bound to enzymes can provide a readout of the electrostatic active site environment, an environment that is otherwise difficult to assess.

    View details for DOI 10.1021/ja026481z

    View details for Web of Science ID 000178104600035

    View details for PubMedID 12236744

  • Probing the Tetrahymena group I ribozyme reaction in both directions BIOCHEMISTRY Karbstein, K., Carroll, K. S., Herschlag, D. 2002; 41 (37): 11171-11183

    Abstract

    The Tetrahymena L-21 ScaI ribozyme derived from the self-splicing group I intron catalyzes a reversible reaction analogous to the first step of self-splicing: CCCUCUA (S) + [UC]G right harpoon over left harpoon CCCUCU (P) + [UC]GA. To relate our understanding of the ribozyme to the self-splicing reaction and to further the mechanistic dissection of the ribozyme reaction, we have established a quantitative kinetic and thermodynamic framework for the forward and reverse reaction of the L-21 ScaI ribozyme under identical conditions. Examination of the framework shows that binding of products is cooperative with binding enhanced 5-fold, as was observed previously for binding of the substrates. Further, binding of UCGA is 12-fold weaker than binding of the unphosphorylated UCG, analogous to the 20-fold weaker binding of phosphorylated S relative to P; the molecular interactions underlying the stronger binding of UCG were traced to the 3'-hydroxyl group of UCG. The symmetrical effects on binding of substrates and products result in the equilibrium between ribozyme-bound species, K(int), that is essentially unperturbed from the solution equilibrium, K(ext) (K(int) = [E.P.UCGA]/[E.S.UCG] = 4.6 and K(ext) = [P][UCGA]/[S][UCG] = 1.9). Last, we show that the pK(a) values of the nucleophiles in the forward and reverse reactions are >/=10. This observation suggests that metal ion activation of the nucleophile and stabilization of the leaving group can only account for a portion of the rate enhancement of this ribozyme. These and prior results suggest that the Tetrahymena group I ribozyme, analogous to protein enzymes, uses multiple catalytic strategies to achieve its large rate enhancement.

    View details for Web of Science ID 000178011700013

    View details for PubMedID 12220182

  • Dissection of a metal-ion-mediated conformational change in Tetrahlymena ribozyme catalysis RNA-A PUBLICATION OF THE RNA SOCIETY Shan, S. O., Herschlag, D. 2002; 8 (7): 861-872

    Abstract

    Conformational changes are often required for the biological function of RNA molecules. In the Tetrahymena group I ribozyme reaction, a conformational change has been suggested to occur upon binding of the oligonucleotide substrate (S) or the guanosine nucleophile (G), leading to stronger binding of the second substrate. Recent work showed that the two substrates are bridged by a metal ion that coordinates both the nonbridging reactive phosphoryl oxygen of S and the 2'-OH of G. These results suggest that the energy from the metal ion substrate interactions is used to drive the proposed conformational change. In this work, we provide an experimental test for this model. The results provide strong support for the proposed conformational change and for a central role of the bridging metal ion in this change. The results from this work, combined with previous data, allow construction of a two-state model that quantitatively accounts for all of the observations in this and previous-work. This model provides a conceptual and quantitative framework that will facilitate understanding and further probing of the energetic and structural features of this conformational change and its role in catalysis.

    View details for DOI 10.1017/S1355838202020216

    View details for Web of Science ID 000176943100002

    View details for PubMedID 12166641

  • Precision and functional specificity in mRNA decay PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wang, Y. L., Liu, C. L., Storey, J. D., Tibshirani, R. J., Herschlag, D., Brown, P. O. 2002; 99 (9): 5860-5865

    Abstract

    Posttranscriptional processing of mRNA is an integral component of the gene expression program. By using DNA microarrays, we precisely measured the decay of each yeast mRNA, after thermal inactivation of a temperature-sensitive RNA polymerase II. The half-lives varied widely, ranging from approximately 3 min to more than 90 min. We found no simple correlation between mRNA half-lives and ORF size, codon bias, ribosome density, or abundance. However, the decay rates of mRNAs encoding groups of proteins that act together in stoichiometric complexes were generally closely matched, and other evidence pointed to a more general relationship between physiological function and mRNA turnover rates. The results provide strong evidence that precise control of the decay of each mRNA is a fundamental feature of the gene expression program in yeast.

    View details for DOI 10.1073/pnas.092538799

    View details for Web of Science ID 000175377800023

    View details for PubMedID 11972065

  • RNA simulations: Probing hairpin unfolding and the dynamics of a GNRA tetraloop JOURNAL OF MOLECULAR BIOLOGY Sorin, E. J., Engelhardt, M. A., Herschlag, D., Pande, V. S. 2002; 317 (4): 493-506

    Abstract

    Simulations of an RNA hairpin containing a GNRA tetraloop were conducted to allow the characterization of its secondary structure formation and dynamics. Ten 10 ns trajectories of the folded hairpin 5'-GGGC[GCAA]GCCU-3' were generated using stochastic dynamics and the GB/SA implicit solvent model at 300 K. Overall, we find the stem to be a very stable subunit of this molecule, whereas multiple loop conformations and transitions between them were observed. These trajectories strongly suggest that extension of the C6 base away from the loop occurs cooperatively with an N-type-->S-type sugar pucker conversion in that residue and that similar pucker transitions are necessary to stabilize other looped-out bases. In addition, a short-lived conformer with an extended fourth loop residue (A8) lacking this stabilizing 2'-endo pucker mode was observed. Results of thermal perturbation at 400 K support this model of loop dynamics. Unfolding trajectories were produced using this same methodology at temperatures of 500 to 700 K. The observed unfolding events display three-state behavior kinetically (including native, globular, and unfolded populations) and, based on these observations, we propose a folding mechanism that consists of three distinct events: (i) collapse of the random unfolded structure and sampling of the globular state; (ii) passage into the folded region of configurational space as stem base-pairs form and gain helicity; and (iii) attainment of proper loop geometry and organization of loop pairing and stacking interactions. These results are considered in the context of current experimental knowledge of this and similar nucleic acid hairpins.

    View details for DOI 10.1006/jmbi.2002.5447

    View details for Web of Science ID 000175070100002

    View details for PubMedID 11955005

  • Rapid compaction during RNA folding PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Russell, R., Millettt, I. S., Tate, M. W., Kwok, L. W., Nakatani, B., Gruner, S. M., Mochrie, S. G., Pande, V., Doniach, S., Herschlag, D., Pollack, L. 2002; 99 (7): 4266-4271

    Abstract

    We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.

    View details for DOI 10.1073/pnas.072589599

    View details for Web of Science ID 000174856000027

    View details for PubMedID 11929997

  • Alkaline phosphatase revisited: Hydrolysis of alkyl phosphates BIOCHEMISTRY O'Brien, P. J., Herschlag, D. 2002; 41 (9): 3207-3225

    Abstract

    Escherichia coli alkaline phosphatase (AP) is the prototypical two metal ion catalyst with two divalent zinc ions bound approximately 4 A apart in the active site. Studies spanning half a century have elucidated many structural and mechanistic features of this enzyme, rendering it an attractive model for investigating the potent catalytic power of bimetallic centers. Unfortunately, fundamental mechanistic features have been obscured by limitations with the standard assays. These assays generate concentrations of inorganic phosphate (P(i)) in excess of its inhibition constant (K(i) approximately 1 muM). This tight binding by P(i) has affected the majority of published kinetic constants. Furthermore, binding limits k(cat)/K(m) for reaction of p-nitrophenyl phosphate, the most commonly employed substrate. We describe a sensitive (32)P-based assay for hydrolysis of alkyl phosphates that avoids the complication of product inhibition. We have revisited basic mechanistic features of AP with these alkyl phosphate substrates. The results suggest that the chemical step for phosphorylation of the enzyme limits k(cat)/K(m). The pH-rate profile and additional results suggest that the serine nucleophile is active in its anionic form and has a pK(a) of < or = 5.5 in the free enzyme. An inactivating pK(a) of 8.0 is observed for binding of both substrates and inhibitors, and we suggest that this corresponds to ionization of a zinc-coordinated water molecule. Counter to previous suggestions, inorganic phosphate dianion appears to bind to the highly charged AP active site at least as strongly as the trianion. The dependence of k(cat)/K(m) on the pK(a) of the leaving group follows a Brønsted correlation with a slope of beta(lg) = -0.85 +/- 0.1, differing substantially from the previously reported value of -0.2 obtained from data with a less sensitive assay. This steep leaving group dependence is consistent with a largely dissociative transition state for AP-catalyzed hydrolysis of phosphate monoesters. The new (32)P-based assay employed herein will facilitate continued dissection of the AP reaction by providing a means to readily follow the chemical step for phosphorylation of the enzyme.

    View details for DOI 10.1021/bi012166y

    View details for Web of Science ID 000174187500035

    View details for PubMedID 11863460

  • Exploring the folding landscape of a structured RNA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Russell, R., Zhuang, X. W., Babcock, H. P., Millett, I. S., Doniach, S., Chu, S., Herschlag, D. 2002; 99 (1): 155-160

    Abstract

    Structured RNAs achieve their active states by traversing complex, multidimensional energetic landscapes. Here we probe the folding landscape of the Tetrahymena ribozyme by using a powerful approach: the folding of single ribozyme molecules is followed beginning from distinct regions of the folding landscape. The experiments, combined with small-angle x-ray scattering results, show that the landscape contains discrete folding pathways. These pathways are separated by large free-energy barriers that prevent interconversion between them, indicating that the pathways lie in deep channels in the folding landscape. Chemical protection and mutagenesis experiments are then used to elucidate the structural features that determine which folding pathway is followed. Strikingly, a specific long-range tertiary contact can either help folding or hinder folding, depending on when it is formed during the process. Together these results provide an unprecedented view of the topology of an RNA folding landscape and the RNA structural features that underlie this multidimensional landscape.

    View details for Web of Science ID 000173233300031

    View details for PubMedID 11756689

  • Role of SRP RNA in the GTPase cycles of ffh and FtsY BIOCHEMISTRY Peluso, P., Shan, S. O., Nock, S., Herschlag, D., Walter, P. 2001; 40 (50): 15224-15233

    Abstract

    The bacterial homologues of the signal recognition particle (SRP) and its receptor, the Ffh*4.5S RNA ribonucleoprotein complex and the FtsY protein, respectively, form a unique complex in which both Ffh and FtsY act as GTPase activating proteins for one another, resulting in the mutual stimulation of GTP hydrolysis by both proteins. Previous work showed that 4.5S RNA enhances the GTPase activity in the presence of both Ffh and FtsY, but it was not clear how this was accomplished. In this work, kinetic and thermodynamic analyses of the GTPase reactions of Ffh and FtsY have provided insights into the role of 4.5S RNA in the GTPase cycles of Ffh and FtsY. We found that 4.5S RNA accelerates the association between Ffh and FtsY 400-fold in their GTP-bound form, analogous to its 200-fold catalytic effect on Ffh*FtsY association previously observed with the GppNHp-bound form [Peluso, P., et al. (2000) Science 288, 1640-1643]. Further, Ffh-FtsY association is rate-limiting for the observed GTPase reaction with subsaturating Ffh and FtsY, thereby accounting for the apparent stimulatory effect of 4.5S RNA on the GTPase activity observed previously. An additional step, GTP hydrolysis from the Ffh*FtsY complex, is also moderately facilitated by 4.5S RNA. These results suggest that 4.5S RNA modulates the conformation of the Ffh*FtsY complex and may, in turn, regulate its GTPase activity during the SRP functional cycle.

    View details for DOI 10.1021/bi011639y

    View details for Web of Science ID 000172723800018

    View details for PubMedID 11735405

  • Probing the folding landscape of the Tetrahymena ribozyme: Commitment to form the native conformation is late in the folding pathway JOURNAL OF MOLECULAR BIOLOGY Russell, R., Herschlag, D. 2001; 308 (5): 839-851

    Abstract

    Large, structured RNAs traverse folding landscapes in which intermediates and long-lived misfolded states are common. To obtain a comprehensive description of the folding landscape for a structured RNA, it is necessary to understand the connections between productive folding pathways and pathways to these misfolded states. The Tetrahymena group I ribozyme partitions between folding to the native state and to a long-lived misfolded conformation. Here, we show that the observed rate constant for commitment to fold to the native or misfolded states is 1.9 min(-1) (37 degrees C, 10 mM Mg(2+)), the same within error as the rate constant for overall folding to the native state. Thus, the commitment to alternative folding pathways is made late in the folding process, concomitant with or after the rate-limiting step for overall folding. The ribozyme forms much of its tertiary structure significantly faster than it reaches this commitment point and the tertiary structure is expected to be stable, suggesting that the commitment to fold along pathways to the native or misfolded states is made from a partially structured intermediate. These results allow the misfolded conformation to be incorporated into a folding framework that reconciles previous data and gives quantitative information about the energetic topology of the folding landscape for this RNA.

    View details for Web of Science ID 000169014500002

    View details for PubMedID 11352576

  • Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of Escherichia coli alkaline phosphatase BIOCHEMISTRY O'Brien, P. J., Herschlag, D. 2001; 40 (19): 5691-5699

    Abstract

    Escherichia coli alkaline phosphatase (AP) is a proficient phosphomonoesterase with two Zn(2+) ions in its active site. Sequence homology suggests a distant evolutionary relationship between AP and alkaline phosphodiesterase/nucleotide pyrophosphatase, with conservation of the catalytic metal ions. Furthermore, many other phosphodiesterases, although not evolutionarily related, have a similar active site configuration of divalent metal ions in their active sites. These observations led us to test whether AP could also catalyze the hydrolysis of phosphate diesters. The results described herein demonstrate that AP does have phosphodiesterase activity: the phosphatase and phosphodiesterase activities copurify over several steps; inorganic phosphate, a strong competitive inhibitor of AP, inhibits the phosphodiesterase and phosphatase activities with the same inhibition constant; a point mutation that weakens phosphate binding to AP correspondingly weakens phosphate inhibition of the phosphodiesterase activity; and mutation of active site residues substantially reduces both the mono- and diesterase activities. AP accelerates the rate of phosphate diester hydrolysis by 10(11)-fold relative to the rate of the uncatalyzed reaction [(k(cat)/K(m))/k(w)]. Although this rate enhancement is substantial, it is at least 10(6)-fold less than the rate enhancement for AP-catalyzed phosphate monoester hydrolysis. Mutational analysis suggests that common active site features contribute to hydrolysis of both phosphate monoesters and phosphate diesters. However, mutation of the active site arginine to serine, R166S, decreases the monoesterase activity but not the diesterase activity, suggesting that the interaction of this arginine with the nonbridging oxygen(s) of the phosphate monoester substrate provides a substantial amount of the preferential hydrolysis of phosphate monoesters. The observation of phosphodiesterase activity extends the previous observation that AP has a low level of sulfatase activity, further establishing the functional interrelationships among the sulfatases, phosphatases, and phosphodiesterases within the evolutionarily related AP superfamily. The catalytic promiscuity of AP could have facilitated divergent evolution via gene duplication by providing a selective advantage upon which natural selection could have acted.

    View details for DOI 10.1021/bi0028892

    View details for Web of Science ID 000168635900013

    View details for PubMedID 11341834

  • Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction BIOCHEMISTRY Shan, S., Kravchuk, A. V., Piccirilli, J. A., Herschlag, D. 2001; 40 (17): 5161-5171

    Abstract

    Divalent metal ions play a crucial role in catalysis by many RNA and protein enzymes that carry out phosphoryl transfer reactions, and defining their interactions with substrates is critical for understanding the mechanism of biological phosphoryl transfer. Although a vast amount of structural work has identified metal ions bound at the active site of many phosphoryl transfer enzymes, the number of functional metal ions and the full complement of their catalytic interactions remain to be defined for any RNA or protein enzyme. Previously, thiophilic metal ion rescue and quantitative functional analyses identified the interactions of three active site metal ions with the 3'- and 2'-substrate atoms of the Tetrahymena group I ribozyme. We have now extended these approaches to probe the metal ion interactions with the nonbridging pro-S(P) oxygen of the reactive phosphoryl group. The results of this study combined with previous mechanistic work provide evidence for a novel assembly of catalytic interactions involving three active site metal ions. One metal ion coordinates the 3'-departing oxygen of the oligonucleotide substrate and the pro-S(P) oxygen of the reactive phosphoryl group; another metal ion coordinates the attacking 3'-oxygen of the guanosine nucleophile; a third metal ion bridges the 2'-hydroxyl of guanosine and the pro-S(P) oxygen of the reactive phosphoryl group. These results for the first time define a complete set of catalytic metal ion/substrate interactions for an RNA or protein enzyme catalyzing phosphoryl transfer.

    View details for Web of Science ID 000168435100007

    View details for PubMedID 11318638

  • Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations RNA-A PUBLICATION OF THE RNA SOCIETY O'Rear, J. L., Wang, S. L., Feig, A. L., Beigelman, L., Uhlenbeck, O. C., Herschlag, D. 2001; 7 (4): 537-545

    Abstract

    Although the hammerhead reaction proceeds most efficiently in divalent cations, cleavage in 4 M LiCl is only approximately 10-fold slower than under standard conditions of 10 mM MgCl2 (Murray et al., Chem Biol, 1998, 5:587-595; Curtis & Bartel, RNA, 2001, this issue, pp. 546-552). To determine if the catalytic mechanism with high concentrations of monovalent cations is similar to that with divalent cations, we compared the activities of a series of modified hammerhead ribozymes in the two ionic conditions. Nearly all of the modifications have similar deleterious effects under both reaction conditions, suggesting that the hammerhead adopts the same general catalytic structure with both monovalent and divalent cations. However, modification of three ligands previously implicated in the binding of a functional divalent metal ion have substantially smaller effects on the cleavage rate in Li+ than in Mg2+. This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.

    View details for Web of Science ID 000168500400004

    View details for PubMedID 11345432

  • Chemical rescue of phosphoryl transfer in a cavity mutant: a cautionary tale for site-directed mutagenesis BIOCHEMISTRY Admiraal, S. J., Meyer, P., Schneider, B., Deville-Bonne, D., Janin, J., Herschlag, D. 2001; 40 (2): 403-413

    Abstract

    We have explored the ability of a nucleoside diphosphate kinase (NDPK) mutant in which the nucleophilic histidine has been replaced by glycine (H122G) to transfer phosphate from ATP to alcohols of varying pK(a), size, shape, and polarity. This cavity mutant does indeed act as a primitive alcohol kinase. The rate of its phosphoryl transfer to alcohols varies considerably, with values spanning a DeltaDeltaG(double dagger) range of 4 kcal/mol, whereas the alcohols have very similar intrinsic reactivities. Analysis of these results suggests that the ability to carry out phosphoryl transfer within the cavity is not a simple function of being small enough to enter the cavity, but rather is a complex function of steric, solvation, entropic, van der Waals packing, and electrostatic properties of the alcohol. In addition, large differences are observed between the reactivities of alcohols within the nucleophile cavity of H122G and the reactivities of the same alcohols within the nucleophile cavity of H122A, a mutant NDPK that differs from H122G by a single methyl group within the cavity. The crystal structures of the two cavity mutants are very similar to one another and to wild-type NDPK, providing no evidence for a structurally perturbed active site. The differences in reactivity between the two mutant proteins illustrate a fundamental limitation of energetic analysis from site-directed mutagenesis: although removal of a side chain is generally considered to be a conservative change, the energetic effects of any given mutation are inextricably linked to the molecular properties of the created cavity and the surrounding protein environment.

    View details for DOI 10.1021/bi002472w

    View details for Web of Science ID 000166496900013

    View details for PubMedID 11148034

  • A single-molecule study of RNA catalysis and folding SCIENCE Zhuang, X. W., Bartley, L. E., Babcock, H. P., Russell, R., Ha, T. J., Herschlag, D., Chu, S. 2000; 288 (5473): 2048-?

    Abstract

    Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules. The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistinguishable from the unmodified free ribozyme in solution. A reversible local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determination of the rate constants and characterization of the transition state. A rarely populated docked state, not measurable by ensemble methods, was observed. In the overall folding process, intermediate folding states and multiple folding pathways were observed. In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered. These results establish single-molecule fluorescence as a powerful tool for examining RNA folding.

    View details for Web of Science ID 000087687000052

    View details for PubMedID 10856219

  • Role of 4.5S RNA in assembly of the bacterial signal recognition particle with its receptor SCIENCE Peluso, P., Herschlag, D., Nock, S., Freymann, D. M., Johnson, A. E., Walter, P. 2000; 288 (5471): 1640-1643

    Abstract

    The mechanism by which a signal recognition particle (SRP) and its receptor mediate protein targeting to the endoplasmic reticulum or to the bacterial plasma membrane is evolutionarily conserved. In Escherichia coli, this reaction is mediated by the Ffh/4.5S RNA ribonucleoprotein complex (Ffh/4.5S RNP; the SRP) and the FtsY protein (the SRP receptor). We have quantified the effects of 4.5S RNA on Ffh-FtsY complex formation by monitoring changes in tryptophan fluorescence. Surprisingly, 4.5S RNA facilitates both assembly and disassembly of the Ffh-FtsY complex to a similar extent. These results provide an example of an RNA molecule facilitating protein-protein interactions in a catalytic fashion.

    View details for Web of Science ID 000087382300040

    View details for PubMedID 10834842

  • An unconventional origin of metal-ion rescue and inhibition in the Tetrahymena group I ribozyme reaction RNA-A PUBLICATION OF THE RNA SOCIETY Shan, S. O., Herschlag, D. 2000; 6 (6): 795-813

    Abstract

    The presence of catalytic metal ions in RNA active sites has often been inferred from metal-ion rescue of modified substrates and sometimes from inhibitory effects of alternative metal ions. Herein we report that, in the Tetrahymena group I ribozyme reaction, the deleterious effect of a thio substitution at the pro-Sp position of the reactive phosphoryl group is rescued by Mn2+. However, analysis of the reaction of this thio substrate and of substrates with other modifications strongly suggest that this rescue does not stem from a direct Mn2+ interaction with the Sp sulfur. Instead, the apparent rescue arises from a Mn2+ ion interacting with the residue immediately 3' of the cleavage site, A(+1), that stabilizes the tertiary interactions between the oligonucleotide substrate (S) and the active site. This metal site is referred to as site D herein. We also present evidence that a previously observed Ca2+ ion that inhibits the chemical step binds to metal site D. These and other observations suggest that, whereas the interactions of Mn2+ at site D are favorable for the chemical reaction, the Ca2+ at site D exerts its inhibitory effect by disrupting the alignment of the substrates within the active site. These results emphasize the vigilance necessary in the design and interpretation of metal-ion rescue and inhibition experiments. Conversely, in-depth mechanistic analysis of the effects of site-specific substrate modifications can allow the effects of specific metal ion-RNA interactions to be revealed and the properties of individual metal-ion sites to be probed, even within the sea of metal ions bound to RNA.

    View details for Web of Science ID 000087503100002

    View details for PubMedID 10864040

  • Use of duplex rigidity for stability and specificity in RNA tertiary structure BIOCHEMISTRY Narlikar, G. J., Bartley, L. E., Herschlag, D. 2000; 39 (20): 6183-6189

    Abstract

    The Tetrahymena group I ribozyme's oligonucleotide substrate, CCCUCUA(5), forms six base pairs with the ribozyme's internal guide sequence (IGS, 5'GGAGGG) to give the P1 duplex, and this duplex then docks into the active site via tertiary interactions. Shortening the substrate by three residues to give UCUA(5) reduces the equilibrium constant for P1 docking by approximately 200-fold even though UCUA(5) retains all the functional groups known to be involved in tertiary interactions [Narlikar, G. J., Bartley, L. E., Khosla, M., and Herschlag, D. (1999) Biochemistry 38, 14192-14204]. Here we show that the P1 duplex formed with UCUA(5) engages in all of the major tertiary interactions made by the standard P1 duplex. This suggests that the destabilization is not due to disruption of specific tertiary interactions. It therefore appears that the weaker docking of UCUA(5) arises from the increased conformational freedom of the undocked P1 duplex, which has three unpaired IGS residues and thus a larger entropic cost for docking. Further, a 2'-methoxy substitution at an IGS residue that is base-paired in the standard P1 duplex with CCCUCUA(5) but unpaired in the P1 duplex with UCUA(5) destabilizes docking of the standard P1 duplex approximately 300-fold more than it destabilizes docking of the P1 duplex formed with UCUA(5). These results suggest that fixation of groups in the context of a rigid duplex may be a general strategy used by RNA to substantially increase interaction specificity, both by aiding binding of the desired functional groups and by increasing the energetic cost of forming alternative interactions.

    View details for Web of Science ID 000087399700031

    View details for PubMedID 10821693

  • Small angle X-ray scattering reveals a compact intermediate in RNA folding NATURE STRUCTURAL BIOLOGY Russell, R., Millett, I. S., Doniach, S., Herschlag, D. 2000; 7 (5): 367-370

    Abstract

    We have used small angle X-ray scattering (SAXS) to monitor changes in the overall size and shape of the Tetrahymena ribozyme as it folds. The native ribozyme, formed in the presence of Mg2+, is much more compact and globular than the ensemble of unfolded conformations. Time-resolved measurements show that most of the compaction occurs at least 20-fold faster than the overall folding to the native state, suggesting that a compact intermediate or family of intermediates is formed early and then rearranges in the slow steps that limit the overall folding rate. These results lead to a kinetic folding model in which an initial 'electrostatic collapse' of the RNA is followed by slower rearrangements of elements that are initially mispositioned.

    View details for Web of Science ID 000086908800009

    View details for PubMedID 10802731

  • The substrate-assisted general base catalysis model for phosphate monoester hydrolysis: Evaluation using reactivity comparisons JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Admiraal, S. J., Herschlag, D. 2000; 122 (10): 2145-2148
  • The P5abc peripheral element facilitates preorganization of the Tetrahymena group I ribozyme for catalysis BIOCHEMISTRY Engelhardt, M. A., Doherty, E. A., Knitt, D. S., Doudna, J. A., Herschlag, D. 2000; 39 (10): 2639-2651

    Abstract

    Phylogenetic comparisons and site-directed mutagenesis indicate that group I introns are composed of a catalytic core that is universally conserved and peripheral elements that are conserved only within intron subclasses. Despite this low overall conservation, peripheral elements are essential for efficient splicing of their parent introns. We have undertaken an in-depth structure-function analysis to investigate the role of one of these elements, P5abc, using the well-characterized ribozyme derived from the Tetrahymena group I intron. Structural comparisons using solution-based free radical cleavage revealed that a ribozyme lacking P5abc (E(DeltaP5abc)) and E(DeltaP5abc) with P5abc added in trans (E(DeltaP5abc).P5abc) adopt a similar global tertiary structure at Mg(2+) concentrations greater than 20 mM [Doherty, E. A., et al. (1999) Biochemistry 38, 2982-90]. However, free E(DeltaP5abc) is greatly compromised in overall oligonucleotide cleavage activity, even at Mg(2+) concentrations as high as 100 mM. Further characterization of E(DeltaP5abc) via DMS modification revealed local structural differences at several positions in the conserved core that cluster around the substrate binding sites. Kinetic and thermodynamic dissection of individual reaction steps identified defects in binding of both substrates to E(DeltaP5abc), with > or =25-fold weaker binding of a guanosine nucleophile and > or =350-fold weaker docking of the oligonucleotide substrate into its tertiary interactions with the ribozyme core. These defects in binding of the substrates account for essentially all of the 10(4)-fold decrease in overall activity of the deletion mutant. Together, the structural and functional observations suggest that the P5abc peripheral element not only provides stability but also positions active site residues through indirect interactions, thereby preferentially stabilizing the active ribozyme structure relative to alternative less active states. This is consistent with the view that peripheral elements engage in a network of mutually reinforcing interactions that together ensure cooperative folding of the ribozyme to its active structure.

    View details for Web of Science ID 000085905300023

    View details for PubMedID 10704214

  • The role of the cleavage site 2 '-hydroxyl in the Tetrahymena group I ribozyme reaction CHEMISTRY & BIOLOGY Yoshida, A., Shan, S., Herschlag, D., Piccirilli, J. A. 2000; 7 (2): 85-96

    Abstract

    The 2'-hydroxyl of U preceding the cleavage site, U(-1), in the Tetrahymena ribozyme reaction contributes 10(3)-fold to catalysis relative to a 2'-hydrogen atom. Previously proposed models for the catalytic role of this 2'-OH involve coordination of a catalytic metal ion and hydrogen-bond donation to the 3'-bridging oxygen. An additional model, hydrogen-bond donation by the 2'-OH to a nonbridging reactive phosphoryl oxygen, is also consistent with previous results. We have tested these models using atomic-level substrate modifications and kinetic and thermodynamic analyses.Replacing the 2'-OH with -NH(3)(+) increases the reaction rate approximately 60-fold, despite the absence of lone-pair electrons on the 2'-NH(3)(+) group to coordinate a metal ion. Binding and reaction of a modified oligonucleotide substrate with 2'-NH(2) at U(-1) are unaffected by soft-metal ions. These results suggest that the 2'-OH of U(-1) does not interact with a metal ion. The contribution of the 2'-moiety of U(-1) is unperturbed by thio substitution at either of the nonbridging oxygens of the reactive phosphoryl group, providing no indication of a hydrogen bond between the 2'-OH and the nonbridging phosphoryl oxygens. In contrast, the 10(3)-fold catalytic advantage of 2'-OH relative to 2'-H is eliminated when the 3'-bridging oxygen is replaced by sulfur. As sulfur is a weaker hydrogen-bond acceptor than oxygen, this effect suggests a hydrogen-bonding interaction between the 2'-OH and the 3'-bridging oxygen.These results provide the first experimental support for the model in which the 2'-OH of U(-1) donates a hydrogen bond to the neighboring 3'-bridging oxygen, thereby stabilizing the developing negative charge on the 3'-oxygen in the transition state.

    View details for Web of Science ID 000085171600004

    View details for PubMedID 10662698

  • Does the active site arginine change the nature of the transition state for alkaline phosphatase-catalyzed phosphoryl transfer? JOURNAL OF THE AMERICAN CHEMICAL SOCIETY O'Brien, P. J., Herschlag, D. 1999; 121 (47): 11022-11023
  • Kinetic dissection of fundamental processes of eukaryotic translation initiation in vitro EMBO JOURNAL Lorsch, J. R., Herschlag, D. 1999; 18 (23): 6705-6717

    Abstract

    Approaches have been developed for the kinetic dissection of eukaryotic translation initiation in vitro using rabbit reticulocyte ribosomes and a crude preparation of initiation factors. These new approaches have allowed the kinetics of formation of the 43S and 80S ribosomal complexes to be followed and have substantially improved the ability to follow formation of the first peptide bond. The results suggest the existence of a new step on the initiation pathway that appears to require at least one additional factor and the hydrolysis of GTP and may prepare the 80S complex for the formation of the first peptide bond. The initial kinetic framework and methods developed herein will allow the properties of individual species along the initiation pathway to be probed further and will facilitate dissection of the mechanistic roles of individual translation factors and their interplay with RNA structural elements.

    View details for Web of Science ID 000084177700012

    View details for PubMedID 10581244

  • Stereospecificity of reactions catalyzed by HIV-1 integrase JOURNAL OF BIOLOGICAL CHEMISTRY Gerton, J. L., Herschlag, D., Brown, P. O. 1999; 274 (47): 33480-33487

    Abstract

    The retroviral integrase catalyzes two successive chemical reactions essential for integration of the retroviral genome into a host chromosome: 3' end processing, in which a dinucleotide is cleaved from each 3' end of the viral DNA; and the integration reaction itself, in which the resulting recessed 3' ends of the viral DNA are joined to the host DNA. We have examined the stereospecificity of human immunodeficiency virus type 1 integrase for phosphorothioate substrates in these reactions and in a third reaction, disintegration, which is macroscopically the reverse of integration. Integrase preferentially catalyzed end processing and integration of a substrate with the (R(p))-phosphorothioate stereoisomer at the reaction center and disintegration of a substrate with an (S(p))-phosphorothiate at the reaction center. These results suggest a model for the architecture of the active site of integrase, and its interactions with key features of the viral and target DNA.

    View details for Web of Science ID 000083745200048

    View details for PubMedID 10559232

  • Characterization of a local folding event of the Tetrahymena group I ribozyme: Effects of oligonucleotide substrate length pH, and temperature on the two substrate binding steps BIOCHEMISTRY Narlikar, G. J., Bartley, L. E., Khosla, M., Herschlag, D. 1999; 38 (43): 14192-14204

    Abstract

    Binding of the Tetrahymena group I ribozyme's oligonucleotide substrate occurs in two steps: P1 duplex formation with the ribozyme's internal guide sequence which forms an "open complex" is followed by docking of the P1 duplex into tertiary interactions within the catalytic core which forms a "closed complex". By systematically varying substrate length, pH, and temperature, we have identified conditions under which P1 duplex formation, P1 docking, or the chemical cleavage step limits the rate of the ribozyme reaction. This has enabled characterization of the individual steps as a function of substrate length, pH, and temperature, leading to several conclusions. (1) The rate constant for formation of the open complex is largely independent of substrate length, pH, and temperature, analogous to that of duplex formation in solution. This extends previous results suggesting that open complex formation entails mainly secondary structure formation and strengthens the view that the second binding step, P1 docking, represents a simple transition from secondary to tertiary structure in the context of an otherwise folded RNA. (2) The temperature dependence of the rate constant for P1 docking yields a negative activation entropy, in contrast to the positive entropy change previously observed for the docking equilibrium. This suggests a model in which tertiary interactions are not substantially formed in the transition state for P1 docking. (3) Shortening the substrate by three residues decreases the equilibrium constant for P1 docking by 200-fold, suggesting that the rigidity imposed by full-length duplex formation facilitates formation of tertiary interactions. (4) Once docked, shortened substrates are cleaved at rates within 3-fold of that for the full-length substrate. Thus, all the active site interactions required to accelerate the chemical cleavage event are maintained with shorter substrates. (5) The rate constant of approximately 10(3) min(-1) obtained for P1 docking is significantly faster than the other steps previously identified in the tertiary folding of this RNA. Nevertheless, P1 docking presumably follows other tertiary folding steps because the P1 duplex docks into a core that is formed only upon folding of the rest of the ribozyme.

    View details for Web of Science ID 000083417800010

    View details for PubMedID 10571993

  • Identification of the hammerhead ribozyme metal ion binding site responsible for rescue of the deleterious effect of a cleavage site phosphorothioate BIOCHEMISTRY Wang, S. L., Karbstein, K., Peracchi, A., Beigelman, L., Herschlag, D. 1999; 38 (43): 14363-14378

    Abstract

    The hammerhead ribozyme crystal structure identified a specific metal ion binding site referred to as the P9/G10.1 site. Although this metal ion binding site is approximately 20 A away from the cleavage site, its disruption is highly deleterious for catalysis. Additional published results have suggested that the pro-R(P) oxygen at the cleavage site is coordinated by a metal ion in the reaction's transition state. Herein, we report a study on Cd(2+) rescue of the deleterious phosphorothioate substitution at the cleavage site. Under all conditions, the Cd(2+) concentration dependence can be accounted for by binding of a single rescuing metal ion. The affinity of the rescuing Cd(2+) is sensitive to perturbations at the P9/G10.1 site but not at the cleavage site or other sites in the conserved core. These observations led to a model in which a metal ion bound at the P9/G10.1 site in the ground state acquires an additional interaction with the cleavage site prior to and in the transition state. A titration experiment ruled out the possibility that a second tight-binding metal ion (< 10 microM) is involved in the rescue, further supporting the single metal ion model. Additionally, weakening Cd(2+) binding at the P9/G10.1 site did not result in the biphasic binding curve predicted from other models involving two metal ions. The large stereospecific thio-effects at the P9/G10.1 and the cleavage site suggest that there are interactions with these oxygen atoms in the normal reaction that are compromised by replacement of oxygen with sulfur. The simplest interpretation of the substantial rescue by Cd(2+) is that these atoms interact with a common metal ion in the normal reaction. Furthermore, base deletions and functional group modifications have similar energetic effects on the transition state in the Cd(2+)-rescued phosphorothioate reaction and the wild-type reaction, further supporting the model that a metal ion bridges the P9/G10.1 and the cleavage site in the normal reaction (i.e., with phosphate linkages rather than phosphorothioate linkages). These results suggest that the hammerhead undergoes a substantial conformational rearrangement to attain its catalytic conformation. Such rearrangements appear to be general features of small functional RNAs, presumably reflecting their structural limitations.

    View details for Web of Science ID 000083417800028

    View details for PubMedID 10572011

  • Three metal ions at the active site of the Tetrahymena group I ribozyme PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shan, S. O., Yoshida, A., Sun, S. G., Piccirilli, J. A., Herschlag, D. 1999; 96 (22): 12299-12304

    Abstract

    Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3'-oxygen of the oligonucleotide substrate and the 3'- and 2'-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA.

    View details for Web of Science ID 000083373000021

    View details for PubMedID 10535916

  • Impaired transition state complementarity in the hydrolysis of O-arylphosphorothioates by protein-tyrosine phosphatases BIOCHEMISTRY Zhang, Y. L., Hollfelder, F., Gordon, S. J., Chen, L., Keng, Y. F., Wu, L., Herschlag, D., Zhang, Z. Y. 1999; 38 (37): 12111-12123

    Abstract

    The hydrolysis of O-arylphosphorothioates by protein-tyrosine phosphatases (PTPases) was studied with the aim of providing a mechanistic framework for the reactions of this important class of substrate analogues. O-arylphosphorothioates are hydrolyzed 2 to 3 orders of magnitude slower than O-aryl phosphates by PTPases. This is in contrast to the solution reaction where phosphorothioates display 10-60-fold higher reactivity than the corresponding oxygen analogues. Kinetic analyses suggest that PTPases utilize the same active site and similar kinetic and chemical mechanisms for the hydrolysis of O-arylphosphorothioates and O-aryl phosphates. Thio substitution has no effect on the affinity of substrate or product for the PTPases. Bronsted analyses suggest that like the PTPase-catalyzed phosphoryl transfer reaction the transition state for the PTPase-catalyzed thiophosphoryl transfer is highly dissociative, similar to that of the corresponding solution reaction. The side chain of the active-site Arg residue forms a bidentate hydrogen bond with two of the terminal phosphate oxygens in the ground state and two of the equatorial oxygens in a transition state analog complex with vanadate [Denu et al. (1996) Proc. Natl. Acad. Sci. USA 93, 2493-2498; Zhang, M. et al. (1997) Biochemistry 36, 15-23; Pannifer et al. (1998) J. Biol. Chem. 273, 10454-10462]. Replacement of the active-site Arg409 in the Yersinia PTPase by a Lys reduces the thio effect by 54-fold, consistent with direct interaction and demonstrating strong energetic coupling between Arg409 and the phosphoryl oxygens in the transition state. These results suggest that the large thio effect observed in the PTPase reaction is the result of inability to achieve precise transition state complementarity in the enzyme active site with the larger sulfur substitution.

    View details for Web of Science ID 000082834800029

    View details for PubMedID 10508416

  • New pathways in folding of the Tetrahymena group I RNA enzyme JOURNAL OF MOLECULAR BIOLOGY Russell, R., Herschlag, D. 1999; 291 (5): 1155-1167

    Abstract

    Previous studies have shown that the earliest detectable step in folding of the Tetrahymena ribozyme is tertiary structure formation of the peripheral element P5abc. This, along with other results, has suggested that P5abc may serve as a scaffold upon which additional tertiary structure is built. Herein we use the onset of oligonucleotide cleavage activity as a readout for native state formation and investigate the effect of P5abc on the rate of folding to the native structure. Despite the early folding of P5abc, its removal to give the E delta P5abc variant decreases the rate of attainment of an active structure less than fivefold (20-100 mM Mg2+, 15-50 degrees C). Furthermore, P5abc added in trans is able to bind the folded E delta P5abc ribozyme and promote oligonucleotide cleavage at least tenfold more rapidly than folding of the wild-type ribozyme, indicating that E delta P5abc does not have to first unfold before productively binding P5abc to form the true native state. This suggests that a state with the overall tertiary structure formed but with P5abc unfolded represents a viable on-pathway intermediate for the wild-type ribozyme. These results provide strong evidence for the existence of two pathways to the native state: in one pathway P5abc forms tertiary structure first, and in another it forms late. The pathway in which P5abc forms first is favored because P5abc can fold quickly and because its tertiary structure is stable in the absence of additional structured elements, not because P5abc formation is required for subsequent folding steps. In the course of these experiments, we also found that most of the ribozyme population does not reach the native state directly under standard conditions in vitro, but instead forms an inactive structure that is stable for hours. Finally, the fraction that does fold to the native state folds with a single rate constant of 1 min-1, suggesting that there are no significantly populated "fast-track" pathways that reach the native state directly by avoiding slow folding steps.

    View details for Web of Science ID 000082445900013

    View details for PubMedID 10518951

  • Effects of oligonucleotide length and atomic composition on stimulation of the ATPase activity of translation initiation factor eIF4A RNA-A PUBLICATION OF THE RNA SOCIETY Peck, M. L., Herschlag, D. 1999; 5 (9): 1210-1221

    Abstract

    Eukaryotic translation initiation factor 4A (elF4A) has been proposed to use the energy of ATP hydrolysis to remove RNA structure in the 5' untranslated region (UTR) of mRNAs, helping the 43S ribosomal complex bind to an mRNA and scan to find the 5'-most AUG initiator codon. We have examined the effect of changing the atomic composition and length of single-stranded oligonucleotides on binding to elF4A and on stimulation of its ATPase activity once bound. Substitution of 2'-OH groups with 2'-H or 2'-OCH3 groups reduces ATPase stimulation at least 100-fold, to background levels, without significantly affecting oligonucleotide affinity. These effects suggest that 2'-OH groups participate in an elF4A conformational change that occurs subsequent to oligonucleotide binding and is required for ATPase stimulation. Replacing nonbridging oxygen atoms in phosphodiester linkages with sulfur atoms to make phosphorothioate linkages has no significant effect on stimulation, while substantially increasing affinity. Extending the length of an RNA oligonucleotide from 4 to approximately 15 nt gradually increases oligonucleotide affinity and ATPase stimulation. Consistent with this observation, the increase in affinity and stimulation provided by phosphorothioate linkages and 2'-OH groups is proportional to the number of these groups present within larger oligonucleotides. Further, changing the position of blocks of phosphorothioate linkages or 2'-OH groups within a larger oligonucleotide does not affect affinity and has only a small effect on stimulation. These observations suggest that numerous interactions between the oligonucleotide and elF4A contribute individually to binding and ATPase stimulation. Nevertheless, significant stimulation is observed with as few as four RNA residues. These properties may allow elF4A to operate within regions of 5' UTRs containing only short stretches of exposed single-stranded RNA. As stimulation increases when longer stretches of single-stranded RNA are available, it is possible that the accessibility of single-stranded RNA in a 5' UTR influences translation efficiency.

    View details for Web of Science ID 000082421700010

    View details for PubMedID 10496222

  • Protonated 2 '-aminoguanosine as a probe of the electrostatic environment of the active site of the Tetrahymena group I ribozyme BIOCHEMISTRY Shan, S. O., Narlikar, G. J., Herschlag, D. 1999; 38 (34): 10976-10988

    Abstract

    We have probed the electrostatic environment of the active site of the Tetrahymena group I ribozyme (E) using protonated 2'-aminoguanosine (), in which the 2'-OH of the guanosine nucleophile (G) is replaced by an group. At low concentrations of divalent metal ion (2 mM Mg(2+)), binds at least 200-fold stronger than G or G(NH)()2, with a dissociation constant of

    View details for Web of Science ID 000082342600008

    View details for PubMedID 10460152

  • Probing the role of metal ions in RNA catalysis: Kinetic and thermodynamic characterization of a metal ion interaction with the 2 '-moiety of the guanosine nucleophile in the Tetrahymena group I ribozyme BIOCHEMISTRY Shan, S. O., Herschlag, D. 1999; 38 (34): 10958-10975

    Abstract

    Deciphering the role of individual metal ions in RNA catalysis is a tremendous challenge, as numerous metal ions coat the charged backbone of a folded RNA. Metal ion specificity switch experiments combined with quantitative analysis may provide a powerful tool for probing specific metal ion-RNA interactions and for delineating the role of individual metal ions among the sea of metal ions bound to RNA. We show herein that Mn(2+) rescues the deleterious effect of replacing the 2'-OH of the guanosine nucleophile (G) by -NH(2) (G(NH)()2) in the reaction catalyzed by the Tetrahymena group I ribozyme (E), and the Mn(2+) concentration dependence suggests that a single metal ion is responsible for rescue. This provides strong evidence for a metal ion interaction with the 2'-moiety of G in this ribozyme (referred to as M(C)), confirming and extending previous results in a bacteriophage group I intron [Sjögren, A.-S., Pettersson, E., Sjöberg, B.-M., and Strömberg, R. (1997) Nucleic Acids Res. 25, 648-654]. Toward understanding the >10(6)-fold catalytic contribution of the 2'-OH of G, we have determined the individual reaction steps affected by M(C) and quantitated these effects. has only a small effect on binding of G(NH)()2 to the free ribozyme or ribozyme.oligonucleotide complexes that lack the reactive phosphoryl group. In contrast, increases the binding of G(NH)()2 to the ribozyme.oligonucleotide substrate (E.S) complex 20-fold and increases the binding of S to the E.G(NH)()2 complex by the same amount. These and other observations suggest that M(C) plays an integral role in the coupled binding of the oligonucleotide substrate and the guanosine nucleophile. This metal ion may be used to align the nucleophile within the active site, thereby facilitating the reaction. Alternatively or in addition, M(C) may act in concert with an additional metal ion to coordinate and activate the 3'-OH of G. Finally, these experiments have also allowed us to probe the properties of this metal ion site and isolate the energetic effects of the interaction of this specific metal ion with the 2'-moiety of G.

    View details for Web of Science ID 000082342600007

    View details for PubMedID 10460151

  • Catalysis of phosphoryl transfer from ATP by amine nucleophiles JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Admiraal, S. J., Herschlag, D. 1999; 121 (25): 5837-5845
  • Nucleophilic activation by positioning in phosphoryl transfer catalyzed by nucleoside diphosphate kinase BIOCHEMISTRY Admiraal, S. J., Schneider, B., Meyer, P., Janin, J., Veron, M., Deville-Bonne, D., Herschlag, D. 1999; 38 (15): 4701-4711

    Abstract

    The nonenzymatic reaction of ATP with a nucleophile to generate ADP and a phosphorylated product proceeds via a dissociative transition state with little bond formation to the nucleophile. Consideration of the dissociative nature of the nonenzymatic transition state leads to the following question: To what extent can the nucleophile be activated in enzymatic phosphoryl transfer? We have addressed this question for the NDP kinase reaction. A mutant form of the enzyme lacking the nucleophilic histidine (H122G) can be chemically rescued for ATP attack by imidazole or other exogenous small nucleophiles. The ATP reaction is 50-fold faster with the wild-type enzyme, which has an imidazole nucleophile positioned for reaction by a covalent bond, than with H122G, which employs a noncovalently bound imidazole nucleophile [(kcat/KM)ATP]. Further, a 4-fold advantage for imidazole positioned in the nucleophile binding pocket created by the mutation is suggested from comparison of the reaction of H122G and ATP with an imidazole versus a water nucleophile, after correction for the intrinsic reactivities of imidazole and water toward ATP in solution. X-ray structural analysis shows no detectable rearrangement of the residues surrounding His 122 upon mutation to Gly 122. The overall rate effect of approximately 10(2)-fold for the covalent imidazole nucleophile relative to water is therefore attributed to positioning of the nucleophile with respect to the reactive phosphoryl group. This is underscored by the more deleterious effect of replacing ATP with AlphaTauPgammaS in the wild-type reaction than in the imidazole-rescued mutant reaction, as follows. For the wild-type, AlphaTauPgammaS presumably disrupts positioning between nucleophile and substrate, resulting in a large thio effect of 300-fold, whereas precise alignment is already disrupted in the mutant because there is no covalent bond to the nucleophile, resulting in a smaller thio effect of 10-fold. In summary, the results suggest a catalytic role for activation of the nucleophile by positioning in phosphoryl transfer catalyzed by NDP kinase.

    View details for Web of Science ID 000079918200006

    View details for PubMedID 10200157

  • Catalytic promiscuity and the evolution of new enzymatic activities CHEMISTRY & BIOLOGY O'Brien, P. J., Herschlag, D. 1999; 6 (4): R91-R105

    Abstract

    Several contemporary enzymes catalyze alternative reactions distinct from their normal biological reactions. In some cases the alternative reaction is similar to a reaction that is efficiently catalyzed by an evolutionary related enzyme. Alternative activities could have played an important role in the diversification of enzymes by providing a duplicated gene a head start towards being captured by adaptive evolution.

    View details for Web of Science ID 000082565000001

    View details for PubMedID 10099128

  • Assembly of an exceptionally stable RNA tertiary interface in a group I ribozyme BIOCHEMISTRY Doherty, E. A., Herschlag, D., Doudna, J. A. 1999; 38 (10): 2982-2990

    Abstract

    Group I intron RNAs contain a core of highly conserved helices flanked by peripheral domains that stabilize the core structure. In the Tetrahymena group I ribozyme, the P4, P5, and P6 helices of the core pack tightly against a three-helix subdomain called P5abc. Chemical footprinting and the crystal structure of the Tetrahymena intron P4-P6 domain revealed that tertiary interactions between these two parts of the domain create an extensive solvent-inaccessible interface. We have examined the formation and stability of this tertiary interface by providing the P5abc segment in trans to a Tetrahymena ribozyme construct that lacks P5abc (EDeltaP5abc). Equilibrium gel shift experiments show that the affinity of the P5abc and EDeltaP5abc RNAs is exceptionally strong, with a Kd of approximately 100 pM at 10 mM MgCl2 (at 37 degrees C). Chemical and enzymatic footprinting shows that the RNAs are substantially folded prior to assembly of the complex. Solvent accessibility mapping reveals that, in the absence of P5abc, the intron RNA maintains a nativelike fold but its active-site helices are not tightly packed. Upon binding of P5abc, the catalytic core becomes more tightly packed through indirect effects of the tertiary interface formation. This two-component system facilitates quantitative examination of individual tertiary contacts that stabilize the folded intron.

    View details for Web of Science ID 000079089300014

    View details for PubMedID 10074350

  • Specificity from steric restrictions in the guanosine binding pocket of a group I ribozyme RNA-A PUBLICATION OF THE RNA SOCIETY Russell, R., Herschlag, D. 1999; 5 (2): 158-166

    Abstract

    The 3' splice site of group I introns is defined, in part, by base pairs between the intron core and residues just upstream of the splice site, referred to as P9.0. We have studied the specificity imparted by P9.0 using the well-characterized L-21 Scal ribozyme from Tetrahymena by adding residues to the 5' end of the guanosine (G) that functions as a nucleophile in the oligonucleotide cleavage reaction: CCCUCUA5 (S) + NNG <--> CCCUCU + NNGA5. UCG, predicted to form two base pairs in P9.0, reacts with a (kcat/KM) value approximately 10-fold greater than G, consistent with previous results. Altering the bases that form P9.0 in both the trinucleotide G analog and the ribozyme affects the specificity in the manner predicted for base-pairing. Strikingly, oligonucleotides incapable of forming P9.0 react approximately 10-fold more slowly than G, for which the mispaired residues are simply absent. The observed specificity is consistent with a model in which the P9.0 site is sterically restricted such that an energetic penalty, not present for G, must be overcome by G analogs with 5' extensions. Shortening S to include only one residue 3' of the cleavage site (CCCUCUA) eliminates this penalty and uniformly enhances the reactions of matched and mismatched oligonucleotides relative to guanosine. These results suggest that the 3' portion of S occupies the P9.0 site, sterically interfering with binding of G analogs with 5' extensions. Similar steric effects may more generally allow structured RNAs to avoid formation of incorrect contacts, thereby helping to avoid kinetic traps during folding and enhancing cooperative formation of the correct structure.

    View details for Web of Science ID 000078505300002

    View details for PubMedID 10024168

  • A new heat shock protein that binds nucleic acids JOURNAL OF BIOLOGICAL CHEMISTRY Korber, P., Zander, T., Herschlag, D., Bardwell, J. C. 1999; 274 (1): 249-256

    Abstract

    We describe the isolation of Hsp15, a new, very abundant heat shock protein that binds to DNA and RNA. Hsp15 is well conserved and related to a number of RNA-binding proteins, including ribosomal protein S4, RNA pseudouridine synthase, and tyrosyl-tRNA synthetase. The region shared between these proteins appears to represent a common, but previously unrecognized, RNA binding motif. Filter binding studies showed that Hsp15 binds to a 17-mer single-stranded RNA with a dissociation constant of 9 microM in 22.5 mM Hepes, pH 7. 0, 5 mM MgCl2. A role of Hsp15 in binding nucleic acids puts this protein into a different functional category from that of many other heat shock proteins that act as molecular chaperones or proteases on protein substrates.

    View details for Web of Science ID 000077900200037

    View details for PubMedID 9867837

  • Hydrogen bonding in enzymatic catalysis: Analysis of energetic contributions ENZYME KINETICS AND MECHANISM, PT E Shan, S. O., Herschlag, D. 1999; 308: 246-276

    Abstract

    Enzymes can provide catalysis by increasing the strengthening of hydrogen bonds to groups undergoing charge rearrangement in the course of reaction relative to the strengthening of the hydrogen bonds in the corresponding solution reactions. This can be accomplished by using hydrogen bond donors and acceptors that are stronger than water and by lowering the effective dielectric relative to that in aqueous solution. We suggest that these electrostatic effects are of general significance in enzymatic catalysis. The effective dielectric is lowered by the overall "rigidity" of the folded enzyme, which facilitates the formation of active site interactions, and by the fixation of active site functional groups within the enzyme x substrate complex. This underscores the fundamental interconnection of catalytic mechanisms in enzymatic catalysis.

    View details for Web of Science ID 000084721200011

    View details for PubMedID 10507008

  • Sulfatase activity of E-coli alkaline phosphatase demonstrates a functional link to arylsulfatases, an evolutionarily related enzyme family JOURNAL OF THE AMERICAN CHEMICAL SOCIETY O'Brien, P. J., Herschlag, D. 1998; 120 (47): 12369-12370
  • Structure-function relationships in the hammerhead ribozyme probed by base rescue RNA-A PUBLICATION OF THE RNA SOCIETY Peracchi, A., Matulic-Adamic, J., Wang, S. L., Beigelman, L., Herschlag, D. 1998; 4 (11): 1332-1346

    Abstract

    We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc NatAcad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.

    View details for Web of Science ID 000076768000002

    View details for PubMedID 9814755

  • A core folding model for catalysis by the hammerhead ribozyme accounts for its extraordinary sensitivity to abasic mutations BIOCHEMISTRY Peracchi, A., Karpeisky, A., Maloney, L., Beigelman, L., Herschlag, D. 1998; 37 (42): 14765-14775

    Abstract

    Introducing abasic nucleotides at each of 13 positions in the conserved core of the hammerhead ribozyme causes a large decrease in the extent of catalysis [Peracchi, A., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 11522]. This extreme sensitivity to structural defects is in contrast to the behavior of protein enzymes and larger ribozymes. Several additional differences in the behavior of the hammerhead relative to that of protein enzymes and larger ribozymes are described herein. The deleterious effects of the abasic mutations are not relieved by lowering the temperature, by increasing the concentration of monovalent or divalent metal ions, or by adding polyamines, in contrast to effects observed with protein enzymes and large RNA enzymes. In addition, the abasic mutations do not significantly weaken substrate binding. These results and previous observations are all accounted for by a "core folding" model in which the stable ground state structure of the hammerhead ribozyme complexed with the substrate is a partially folded state that must undergo an additional folding event to achieve its catalytic conformation. We propose that the peculiar behavior of the hammerhead arises because the limited structural interconnections in a small RNA enzyme do not allow the ground state to stably adopt the catalytic conformation; within the globally folded catalytic conformation, limited structural interconnections may further impair catalysis by hampering the precise alignment of active site functional groups. This behavior represents a basic manifestation of the well-recognized interconnection between folding and catalysis.

    View details for Web of Science ID 000076727900013

    View details for PubMedID 9778351

  • RNA structure - Ribozyme crevices and catalysis NATURE Herschlag, D. 1998; 395 (6702): 548-549

    View details for Web of Science ID 000076362900025

    View details for PubMedID 9783578

  • Direct demonstration of the catalytic role of binding interactions in an enzymatic reaction BIOCHEMISTRY Narlikar, G. J., Herschlag, D. 1998; 37 (28): 9902-9911

    Abstract

    It has been suggested that the fundamental feature that distinguishes enzymes from simple chemical catalysts is the ability of enzymes to use binding interactions for catalysis. Results with the Tetrahymena group I RNA enzyme described herein directly demonstrate the catalytic contributions of binding interactions. With wild-type ribozyme, specific functional groups at a distance from the site of chemical transformation facilitate substrate binding without accelerating reaction of bound substrate; with modified ribozymes, these functional groups provide the same overall energetic effect but instead accelerate reaction of bound substrate without increasing binding. These observations are quantitatively described by a structural framework that was established by previous results. The P1 duplex between the substrate and the ribozyme's recognition sequence exists in two states, the open complex, in which the substrate is localized to the ribozyme solely by base-pairing interactions, or the closed complex, in which the duplex is docked into tertiary interactions and positioned with respect to the catalytic groups in the active site. In the absence of sufficient binding energy to ensure stable docking in the ground state, added P1 functional groups accelerate reaction of the bound substrate by helping to overcome the energetic barrier for docking into the reactive, closed complex. When the functional groups present on the P1 duplex are sufficient to ensure stable docking in the closed complex, added functional groups give stronger binding without accelerating reaction of the bound substrate. This behavior is a manifestation of the inextricable link between binding interactions and catalysis. The conclusions also have implications for interpreting the effects of site-directed mutagenesis and for the evolution of active site interactions.

    View details for Web of Science ID 000074893800006

    View details for PubMedID 9665695

  • The DEAD box protein eIF4A. 2. A cycle of nucleotide and RNA-dependent conformational changes BIOCHEMISTRY Lorsch, J. R., Herschlag, D. 1998; 37 (8): 2194-2206

    Abstract

    Limited proteolysis experiments have been carried out with the DEAD box protein eIF4A. The results suggest that there is a substantial conformational change in eIF4A upon binding single-stranded RNA. Binding of ADP induces conformational changes in the free enzyme and the enzyme.RNA complex, and binding of the ATP analogue AMP-PNP induces a conformational change in the enzyme.RNA complex. The presence or absence of the gamma-phosphate on the bound nucleotide acts as a switch, presumably via the Walker motifs, that mediates changes in protein conformation and, as described in the preceding paper in this issue, also mediates changes in RNA affinity. Thus, these results suggest that there is a series of changes in conformation and substrate affinity throughout the ATP hydrolysis reaction cycle. A model is proposed in which eIF4A and the eIF4A-like domains of the DEAD box proteins act as ATP-driven conformational switches or motors that produce movements or structural rearrangements of attached protein domains or associated proteins. These movements could then be used to rearrange RNA structures or RNA.protein complexes.

    View details for Web of Science ID 000072299900013

    View details for PubMedID 9485365

  • The DEAD box protein eIF4A. 1. A minimal kinetic and thermodynamic framework reveals coupled binding of RNA and nucleotide BIOCHEMISTRY Lorsch, J. R., Herschlag, D. 1998; 37 (8): 2180-2193

    Abstract

    eIF4A is the archetypal member of the DEAD box family of proteins and has been proposed to use the energy from ATP hydrolysis to unwind structures in the 5'-untranslated regions of eukaryotic mRNAs during translation initiation. As a step toward understanding the mechanism of action of this class of enzymes, a minimal kinetic and thermodynamic framework for the RNA-activated ATPase function has been established for eIF4A. The enzyme's affinity for ssRNA is modulated by the binding of ATP.Mg2+ and ADP.Mg2+: the affinity of the E.ATP complex for ssRNA is approximately 40-fold higher than that of the E.ADP complex. The enzyme binds its substrates in a random manner; contrary to previous suggestions, neither ATP binding nor hydrolysis is required for binding of single-stranded RNA. The presence or absence of the gamma-phosphate on the bound nucleotide acts as a switch that modulates the enzyme's structure and ssRNA affinity. The data presented in this and the following paper in this issue suggest that ATP binding and hydrolysis produce a cycle of conformational and RNA affinity changes in eIF4A. Such cycles may be used by DEAD box proteins to transduce the energy from ATP hydrolysis into physical work, thereby allowing each member of this family to rearrange its RNA or RNA.protein target.

    View details for Web of Science ID 000072299900012

    View details for PubMedID 9485364

  • Involvement of a specific metal ion in the transition of the hammerhead ribozyme to its catalytic conformation JOURNAL OF BIOLOGICAL CHEMISTRY Peracchi, A., Beigelman, L., Scott, E. C., Uhlenbeck, O. C., Herschlag, D. 1997; 272 (43): 26822-26826

    Abstract

    Previous crystallographic and biochemical studies of the hammerhead ribozyme suggest that a metal ion is ligated by the pro-Rp oxygen of phosphate 9 and by N7 of G10.1 and has a functional role in the cleavage reaction. We have tested this model by examining the cleavage properties of a hammerhead containing a unique phosphorothioate at position 9. The Rp-, but not Sp-, phosphorothioate reduces the cleavage rate by 10(3)-fold, and the rate can be fully restored by addition of low concentrations of Cd2+, a thiophilic metal ion. These results strongly suggest that this bound metal ion is critical for catalysis, despite its location approximately 20 A from the cleavage site in the crystal structure. Analysis of the concentration dependence suggests that Cd2+ binds with a Kd of 25 microM in the ground state and a Kd of 2.5 nM in the transition state. The much stronger transition state binding suggests that the P9 metal ion adopts at least one additional ligand in the transition state and that this metal ion may participate in a large scale conformational change that precedes hammerhead cleavage.

    View details for Web of Science ID A1997YC65900008

    View details for PubMedID 9341112

  • Use of intrinsic binding energy for catalysis by an RNA enzyme PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hertel, K. J., Peracchi, A., Uhlenbeck, O. C., Herschlag, D. 1997; 94 (16): 8497-8502

    Abstract

    The contribution of several individual ribozyme.substrate base pairs to binding and catalysis has been investigated using hammerhead ribozyme substrates that were truncated at their 3' or 5' ends. The base pairs at positions 1.1-2.1 and 15.2-16.2, which flank the conserved core, each contribute 10(4)-fold in the chemical step, without affecting substrate binding. In contrast, base pairs distal to the core contribute to substrate binding but have no effect on the chemical step. These results suggest a "fraying model" in which each ribozyme.substrate helix can exist in either an unpaired ("open") state or a helical ("closed") state, with the closed state required for catalysis. The base pairs directly adjacent to the conserved core contribute to catalysis by allowing the closed state to form. Once the number of base pairs is sufficient to ensure that the closed helical state predominates, additional residues provide stabilization of the helix, and therefore increase binding, but have no further effect on the chemical step. Remarkably, the >5 kcal/mol free energy contribution to catalysis from each of the internal base pairs is considerably greater than the free energy expected for formation of a base pair. It is suggested that this unusually large energetic contribution arises because free energy that is typically lost in constraining residues within a base pair is expressed in the transition state, where it is used for positioning. This extends the concept of "intrinsic binding energy" from protein to RNA enzymes, suggesting that intrinsic binding energy is a fundamental feature of biological catalysis.

    View details for Web of Science ID A1997XQ12400038

    View details for PubMedID 9238005

  • Effects of divalent metal ions on individual steps of the Tetrahymena ribozyme reaction BIOCHEMISTRY McConnell, T. S., Herschlag, D., Cech, T. R. 1997; 36 (27): 8293-8303

    Abstract

    The Tetrahymena thermophila L-21 ScaI ribozyme utilizes Mg2+ to catalyze a site-specific endonuclease reaction analogous to the first step of self-splicing. To better understand the contribution of Mg2+ to ribozyme activity, the Mg2+ concentration dependence of individual rate constants was examined at concentrations greater than those required for ribozyme folding (>2 mM; at 50 degrees C and pH 6.7). Analysis of metal ion inhibition of the chemical step of the reaction indicated that two Ca2+ ions compete with two Mg2+ ions involved in active site chemistry. These Mg2+ ions are bound tightly to the E.S complex (Kd < 2 mM). The rate constant for association of the oligoribonucleotide substrate (S) increased 12-fold from 2 to 100 mM Mg2+ and exhibited saturation behavior, consistent with a single Mg2+ ion involved in S association that binds to the free ribozyme with a Kd for Mg2+ of 15 mM. The preference for the divalent metal ion (Mg2+ congruent with Ca2+ > Ba2+ > Sr2+) suggested that enhancing the rate constant of S association is not simply a function of ionic strength, but is due to a distinct metal ion binding site. Even though Ca2+ does not support reaction, the RNA substrate S was able to bind in the presence of Ca2+. Upon addition of Mg2+, S was cleaved without first dissociating. A model is proposed in which the inactive Ca2+ form of E.S is structurally equivalent to the open complex along the reaction pathway, which has the RNA substrate bound but not docked into the active site. Weaker binding of S in Ca2+ was shown to result from an increase in the rate constant of S dissociation, leading to the proposal that a tight Mg2+ binding site or sites in the E.S complex contribute to the strong binding of S. In summary, the data provide evidence for four functions for bound Mg2+ ions in the catalytic cycle: one increases the rate of RNA substrate binding, one or more decrease the rate of dissociation of S, and two are involved in the chemical step.

    View details for Web of Science ID A1997XK09400013

    View details for PubMedID 9204875

  • Quantitating tertiary binding energies of 2' OH groups on the P1 duplex of the Tetrahymena ribozyme: Intrinsic binding energy in an RNA enzyme BIOCHEMISTRY Narlikar, G. J., Khosla, M., Usman, N., Herschlag, D. 1997; 36 (9): 2465-2477

    Abstract

    Binding of the Tetrahymena ribozyme's oligonucleotide substrate (S) involves P1 duplex formation with the ribozyme's internal guide sequence (IGS) to give an open complex, followed by docking of the P1 duplex into the catalytic core via tertiary interactions to give a closed complex. The overall binding energies provided by 2' OH groups on S and IGS have been measured previously. To obtain the energetic contribution of each of these 2' OH groups in the docking step, we have separately measured their contribution to the stability of a model P1 duplex using "substrate inhibition". This new approach allows measurement of duplex stabilities under conditions identical to those used for ribozyme binding measurements. The tertiary binding energies from the individual 2' OH groups include a small destabilizing contribution of 0.7 kcal/mol and stabilizing contributions of up to -2.9 kcal/mol. The energetic contributions of specific 2' OH groups are discussed in the context of considerable previous work that has characterized the tertiary interactions of the P1 duplex. A "threshold" model for the open and closed complexes is presented that provides a framework to interpret the energetic effects of functional group substitutions on the P1 duplex. The sum of the tertiary stabilization provided by the conserved G x U wobble at the cleavage site and the individual 2' OH groups on the P1 duplex is significantly greater than the observed tertiary stabilization of S (11.0 vs 2.2 kcal/mol). It is suggested that there is an energetic cost for docking the P1 duplex into the active site that is paid for by the "intrinsic binding energy" of groups on the P1 duplex. Substrates that lack sufficient tertiary binding energy to overcome this energetic barrier exhibit reduced reactivities. Thus, the ribozyme appears to use the intrinsic binding energy of groups on the P1 duplex for catalysis. This intrinsic binding energy may be used to position reactants within the active site and to induce electrostatic destabilization of the substrate, relative to its interactions in solution.

    View details for Web of Science ID A1997WL64300014

    View details for PubMedID 9054551

  • Mechanistic aspects of enzymatic catalysis: Lessons from comparison of RNA and protein enzymes ANNUAL REVIEW OF BIOCHEMISTRY Narlikar, G. J., Herschlag, D. 1997; 66: 19-59

    Abstract

    A classic approach in biology, both organismal and cellular, is to compare morphologies in order to glean structural and functional commonalities. The comparative approach has also proven valuable on a molecular level. For example, phylogenetic comparisons of RNA sequences have led to determination of conserved secondary and even tertiary structures, and comparisons of protein structures have led to classifications of families of protein folds. Here we take this approach in a mechanistic direction, comparing protein and RNA enzymes. The aim of comparing RNA and protein enzymes is to learn about fundamental physical and chemical principles of biological catalysis. The more recently discovered RNA enzymes, or ribozymes, provide a distinct perspective on long-standing questions of biological catalysis. The differences described in this review have taught us about the aspects of RNA and proteins that are distinct, whereas the common features have helped us to understand the aspects that are fundamental to biological catalysis. This has allowed the framework that was put forth by Jencks for protein catalysts over 20 years ago (1) to be extended to RNA enzymes, generalized, and strengthened.

    View details for Web of Science ID A1997XH20100003

    View details for PubMedID 9242901

  • The change in hydrogen bond strength accompanying charge rearrangement: Implications for enzymatic catalysis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shan, S. O., Herschlag, D. 1996; 93 (25): 14474-14479

    Abstract

    The equilibrium for formation of the intramolecular hydrogen bond (KHB) in a series of substituted salicylate monoanions was investigated as a function of delta pKa, the difference between the pKa values of the hydrogen bond donor and acceptor, in both water and dimethyl sulfoxide. The dependence of log KHB upon delta pKa is linear in both solvents, but is steeper in dimethyl sulfoxide (slope = 0.73) than in water (slope = 0.05). Thus, hydrogen bond strength can undergo substantially larger increases in nonaqueous media than aqueous solutions as the charge density on the donor or acceptor atom increases. These results support a general mechanism for enzymatic catalysis, in which hydrogen bonding to a substrate is strengthened as charge rearranges in going from the ground state to the transition state; the strengthening of the hydrogen bond would be greater in a nonaqueous enzymatic active site than in water, thus providing a rate enhancement for an enzymatic reaction relative to the solution reaction. We suggest that binding energy of an enzyme is used to fix the substrate in the low-dielectric active site, where the strengthening of the hydrogen bond in the course of a reaction is increased.

    View details for Web of Science ID A1996VY44800053

    View details for PubMedID 8962076

  • Rescue of abasic hammerhead ribozymes by exogenous addition of specific bases PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Peracchi, A., Beigelman, L., Usman, N., Herschlag, D. 1996; 93 (21): 11522-11527

    Abstract

    We have synthesized 13 hammerhead ribozyme variants, each containing an abasic residue at a specific position of the catalytic core. The activity of each of the variants is significantly reduced. In four cases, however, activity can be rescued by exogenous addition of the missing base. For one variant, the rescue is 300-fold; for another, the rescue is to the wild-type level. This latter abasic variant (G10.1X) has been characterized in detail. Activation is specific for guanine, the base initially removed. In addition, the specificity for guanine versus adenine is substantially altered by replacing C with U in the opposite strand of the ribozyme. These results show that a binding site for a small, noncharged ligand can be created in a preexisting ribozyme structure. This has implications for structure-function analysis of RNA, and leads to speculations about evolution in an "RNA world" and about the potential therapeutic use of ribozymes.

    View details for Web of Science ID A1996VM68100042

    View details for PubMedID 8876168

  • Ras-catalyzed hydrolysis of GTP: A new perspective from model studies PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Maegley, K. A., Admiraal, S. J., Herschlag, D. 1996; 93 (16): 8160-8166

    Abstract

    Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach to this problem: the uncatalyzed hydrolysis of GTP is analyzed, and the understanding derived is applied to the Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from this chemical perspective suggests that proton abstraction from the attacking water by a general base and stabilization of charge development on the gamma-phosphoryl oxygen atoms would not be catalytic. Rather, this analysis focuses attention on the GDP leaving group, including the beta-gamma bridge oxygen of GTP, the atom that undergoes the largest change in charge in going from the ground state to the transition state. This leads to a new catalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-nonbridging phosphoryl oxygens and a network of interactions that positions the nucleophilic water molecule and gamma-phosphoryl group with respect to one another may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the beta-gamma bridge oxygen with an Arg side chain that is provided in trans by GAP. The conclusions for Ras and related G proteins are expected to apply more widely to other enzymes that catalyze phosphoryl (-PO(3)2-) transfer, including kinases and phosphatases.

    View details for Web of Science ID A1996VB32500003

    View details for PubMedID 8710841

  • Isolation of a local tertiary folding transition in the context of a globally folded RNA NATURE STRUCTURAL BIOLOGY Narlikar, G. J., Herschlag, D. 1996; 3 (8): 701-710

    Abstract

    Binding of the Tetrahymena ribozyme's oligonucleotide substrate represents a local folding event in the context of a globally folded RNA. Substrate binding involves P1 duplex formation with the ribozyme's internal guide sequence to give an "open complex', followed by docking of the P1 duplex into tertiary interactions to give a "closed complex'. We have isolated the open complex as a thermodynamically stable species using a site-specific modification and high Na+ concentrations. This has allowed characterization of P1 docking, which represents a folding transition between local secondary and local tertiary structure. P1 docking is entropically driven, possibly accompanied by a release of bound water molecules. Strategies analogous to those described here can be used more generally to study local folding events in large structured RNAs and to explore the structural and energetic landscape for RNA folding.

    View details for Web of Science ID A1996VA15000015

    View details for PubMedID 8756329

  • Specificity of hammerhead ribozyme cleavage EMBO JOURNAL Hertel, K. J., Herschlag, D., Uhlenbeck, O. C. 1996; 15 (14): 3751-3757

    Abstract

    To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths.

    View details for Web of Science ID A1996UY92200029

    View details for PubMedID 8670879

  • Energetic effects of multiple hydrogen bonds. Implications for enzymatic catalysis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Shan, S. O., Herschlag, D. 1996; 118 (24): 5515-5518
  • Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: Insights into catalysis and the second step of self-splicing BIOCHEMISTRY Mei, R., Herschlag, D. 1996; 35 (18): 5796-5809

    Abstract

    Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3'-terminal G414 residue of the intron binds. The first step has been extensively characterized in studies of the L-21ScaI ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3'-terminal G414 docked into the G-binding site. This docking is destabilized by approximately 100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K(int) = 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5' exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction.

    View details for Web of Science ID A1996UK01500023

    View details for PubMedID 8639540

  • The energetics of hydrogen bonds in model systems: Implications for enzymatic catalysis SCIENCE Shan, S. O., Loh, S., Herschlag, D. 1996; 272 (5258): 97-101

    Abstract

    Low-barrier or short, strong hydrogen bonds have been proposed to contribute 10 to 20 kilocalories per mole to transition-state stabilization in enzymatic catalysis. The proposal invokes a large increase in hydrogen bond energy when the pKa values of the donor and acceptor (where Ka is the acid constant) become matched in the transition state (delta pKa=0). This hypothesis was tested by investigating the energetics of hydrogen bonds as a function of delta pKa for homologous series of compounds under nonaqueous conditions that are conducive to the formation of low-barrier hydrogen bonds. In all cases, there was a linear correlation between the increase in hydrogen-bond energy and the decrease in delta pKa, as expected from simple electrostatic effects. However, no additional energetic contribution to the hydrogen bond was observed at delta pKa=0. These results and those of other model studies suggest alternative mechanisms by which hydrogen bonds can contribute to enzymatic catalysis, in accord with conventional electrostatic considerations.

    View details for Web of Science ID A1996UD59700046

    View details for PubMedID 8600542

  • pH dependencies of the Tetrahymena ribozyme reveal an unconventional origin of an apparent pK(a) BIOCHEMISTRY Knitt, D. S., Herschlag, D. 1996; 35 (5): 1560-1570

    Abstract

    The L-21 ScaI ribozyme derived from the Tetrahymena thermophila pre-rRNA group I intron catalyzes a site-specific endonucleolytic cleavage of RNA, DNA, and chimeric RNA/DNA oligonucleotides: CCCUCUA5 + G-->CCCUCU + GA5. The pH-rate dependence was determined for the reaction of the E.G complex with the oligonucleotide substrate d(CCCUC)r(U)d(A5) [(kcat/Km)S conditions]. Although it was shown that the pH dependence is not affected by specific buffers, there is inhibition by specific monovalent cations. The intrinsic pH-rate dependence is log-linear with slope 1 below pH 7, displays an apparent pKa of 7.6, remains nearly level until pH 8.5, and then begins to fall. Two models to explain the apparent pKa were ruled out: (1) the pKa represents loss of a proton from the nucleophilic 3' OH of G, and (2) the pKa arises from a change in rate-limiting step from a pH-dependent to a pH-independent step. In addition, these models, or others involving a single titration, cannot account for the decrease in activity at high pH. A third, unconventional, model is consistent with all of the data. It involves inactivation of the ribozyme by any of several independent titrations of groups with pKa values considerably higher than the apparent pKa of 7.6. The data are consistent with loss of catalytic function upon release of a proton from any one of 19 independent sites with pKa = 9.4 (the unperturbed pKa of N1 of G and N3 of U in solution). Independent experiments investigating the effect of pH on different reaction steps supported this model and suggested the identity of some of the required protons. This mechanism of inactivation is expected to generally affect the behavior of RNAs at pH values removed from the pKa of the titrating bases.

    View details for Web of Science ID A1996TU58600027

    View details for PubMedID 8634287

  • MAPPING THE TRANSITION-STATE FOR ATP HYDROLYSIS - IMPLICATIONS FOR ENZYMATIC CATALYSIS CHEMISTRY & BIOLOGY Admiraal, S. J., Herschlag, D. 1995; 2 (11): 729-739

    Abstract

    Phosphoryl transfer, typically involving high energy phosphate donors such as ATP, is the most common class of biological reactions. Despite this, the transition state for phosphoryl transfer from ATP in solution has not been systematically investigated. Characterization of the transition state for the uncatalyzed hydrolysis of ATP would provide a starting point for dissection of enzyme-catalyzed reactions.We examined phosphoryl transfer from ATP, GTP and pyrophosphate to a series of alcohols; these reactions are analogous to the phosphorylation of sugars and other biological alcohols and to the hydrolysis of ATP. The Brønsted beta(nucleophile) value of 0.07 is small, indicating that there is little bond formation between the incoming nucleophile and the electrophilic phosphoryl group in the transition state. Coordination of Mg2+ has no measurable effect on this value. The Brønsted beta(leaving group) value of -1.1 for phosphoryl transfer to water from a series of phosphoanhydrides is large and negative, suggesting that the bond between phosphorous and the leaving group oxygen is largely broken in the transition state.Uncatalyzed hydrolysis of ATP in solution occurs via a dissociative, metaphosphate-like transition state, with little bond formation between nucleophile and ATP and substantial cleavage of the bond between the gamma-phosphoryl moiety and the ADP leaving group. Bound Mg2+ does not perturb the dissociative nature of the transition state, contrary to proposals that enzyme-bound metal ions alter this structure. The simplest expectation for phosphoryl transfer at the active site of enzymes thus entails a dissociative transition state. These results provide a basis for analyzing catalytic mechanisms for phosphoryl transfer.

    View details for Web of Science ID A1995TG85900005

    View details for PubMedID 9383480

  • THE NATURE OF THE TRANSITION-STATE FOR ENZYME-CATALYZED PHOSPHORYL TRANSFER - HYDROLYSIS OF O-ARYL PHOSPHOROTHIOATES BY ALKALINE-PHOSPHATASE BIOCHEMISTRY Hollfelder, F., Herschlag, D. 1995; 34 (38): 12255-12264

    Abstract

    There has been much speculation that enzymes change the nature of the transition state for phosphoryl transfer from the dissociative transition state observed in solution reactions to an associative transition state at the enzyme's active site. This proposal can be tested by comparing linear free energy relationships (LFERs) for nonenzymatic and enzymatic reactions, provided that the specificity of the enzyme's binding site does not perturb the dependence of rate on the intrinsic reactivity of a series of substrates. The shallow binding groove of Escherichia coli alkaline phosphatase (AP) and its wide specificity suggest that this enzyme may be suited for such an approach. A second requirement of this approach is that the actual chemical step is rate-limiting. Comparisons of the reactions of aryl phosphorothioates and aryl phosphates support the previous conclusion that a nonchemical step limits kcat/KM for reactions of aryl phosphates, but suggest that the chemical cleavage step is rate-limiting for the aryl phosphorothioates. We therefore determined the dependence of the rate of AP-catalyzed cleavage of a series of aryl phosphorothioates on the intrinsic reactivity of the substrates. The large negative values of beta leaving group = -0.8 for the enzymatic reaction (kcat/KM) and -1.1 for the nonenzymatic hydrolysis reaction suggest that there is considerable dissociative character in both the enzymatic and nonenzymatic transition states. Despite the wide specificity of AP, certain substrates deviate from the LFER, underscoring that extreme care is required in applying LFERs to enzymatic reactions. The large negative value of beta leaving group suggests that AP can achieve substantial catalysis via a transition state with dissociative character.

    View details for Web of Science ID A1995RX23500021

    View details for PubMedID 7547968

  • RNA CHAPERONES AND THE RNA FOLDING PROBLEM JOURNAL OF BIOLOGICAL CHEMISTRY Herschlag, D. 1995; 270 (36): 20871-20874

    View details for Web of Science ID A1995RU05400001

    View details for PubMedID 7545662

  • USE OF BINDING-ENERGY BY AN RNA ENZYME FOR CATALYSIS BY POSITIONING AND SUBSTRATE DESTABILIZATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Narlikar, G. J., Gopalakrishnan, V., McConnell, T. S., Usman, N., Herschlag, D. 1995; 92 (9): 3668-3672

    Abstract

    A fundamental catalytic principle for protein enzymes in the use of binding interactions away from the site of chemical transformation for catalysis. We have compared the binding and reactivity of a series of oligonucleotide substrates and products of the Tetrahymena ribozyme, which catalyzes a site-specific phosphodiester cleavage reaction: CCCUCUpA+G<-->CCCUCU-OH+GpA. The results suggest that this RNA enzyme, like protein enzymes, can utilize binding interactions to achieve substantial catalysis via entropic fixation and substrate destabilization. The stronger binding of the all-ribose oligonucleotide product compared to an analog with a terminal 3' deoxyribose residue gives an effective concentration of 2200 M for the 3' hydroxyl group, a value approaching those obtained with protein enzymes and suggesting the presence of a structurally well defined active site capable of precise positioning. The stabilization from tertiary binding interactions is 40-fold less for the oligonucleotide substrate than the oligonucleotide product, despite the presence of the reactive phosphoryl group in the substrate. This destabilization is accounted for by a model in which tertiary interactions away from the site of bond cleavage position the electron-deficient 3' bridging phosphoryl oxygen of the oligonucleotide substrate next to an electropositive Mg ion. As the phosphodiester bond breaks and this 3' oxygen atom develops a negative charge in the transition state, the weak interaction of the substrate with Mg2+ becomes strong. These strategies of "substrate destabilization" and "transition state stabilization" provide estimated rate enhancements of approximately 280- and approximately 60-fold, respectively. Analogous substrate destabilization by a metal ion or hydrogen bond donor may be used more generally by RNA and protein enzymes catalyzing reactions of phosphate esters.

    View details for Web of Science ID A1995QV93000007

    View details for PubMedID 7731962

  • DISSECTION OF THE ROLE OF THE CONSERVED G-CENTER-DOT-U PAIR IN GROUP-I RNA SELF-SPLICING BIOCHEMISTRY Knitt, D. S., Narlikar, G. J., Herschlag, D. 1994; 33 (46): 13864-13879

    Abstract

    Phylogenetic conservation among > 100 group I introns and previous in vitro studies have implicated a G.U pair as defining the 5'-splice site for exon ligation. The U residue defines the 3' end of the 5' exon, and the complementary G residue is part of the internal guide sequence (IGS) that base pairs to the 5' exon. We now quantitate the effect of this pair on individual reaction steps using the L-21ScaI ribozyme, which is derived from the group I intron of Tetrahymena thermophila pre-rRNA. The following results indicate that interactions with this G.U pair contribute to the binding of the 5'-exon, the positioning of the 5'-splice site with respect to the catalytic site, and the chemical step. The oligonucleotide, CCCUCU, binds to the ribozyme approximately 20-fold stronger than CCCUCC despite the fact that the U-containing oligonucleotide forms an approximately 5-fold less stable duplex with an oligonucleotide analog of the IGS, GGAGGG. This and two independent experimental observations indicate that the G.U pair contributes approximately 100-fold (3 kcal/mol, 50 degrees C) to tertiary interactions that allow the P1 duplex, which is formed between the 5'-exon and the IGS, to dock into the ribozyme's core. The approximately 50-80-fold increase in miscleavage of 5'-exon analogs upon replacement of the 3'-terminal U of CCCUCU with C or upon removal of the 3'-terminal U suggests that the tertiary interactions with the G.U pair not only contribute to docking but also ensure correct positioning of the 5'-splice site with respect to the catalytic site, thereby minimizing the selection of incorrect splice sites. Comparison of the rates of the chemical cleavage step with G.U vs G.C suggests that the G.U pair contributes approximately 10-fold to the chemical step. It was previously suggested that the 2'-hydroxyl of this U residue helps stabilize the 3'-oxyanion leaving group in the chemical transition state via an intramolecular hydrogen bond. Relative reactivities of oligonucleotide substrates with ribose and deoxyribose U and C are consistent with a model based on a recent X-ray crystallographic structure in which the exocyclic amino group of G helps orient the 2'-hydroxyl of U via a bridging water molecule, thereby strengthening the hydrogen bond donated from the 2'-hydroxyl group to the neighboring incipient 3'-oxyanion. Finally, kinetic and thermodynamic evidence for the formation of a G.C+ wobble pair is presented.(ABSTRACT TRUNCATED AT 400 WORDS)

    View details for Web of Science ID A1994PT48500041

    View details for PubMedID 7947795

  • ESCHERICHIA-COLI PROTEINS, INCLUDING RIBOSOMAL-PROTEIN S12, FACILITATE IN-VITRO SPLICING OF PHAGE-T4 INTRONS BY ACTING AS RNA CHAPERONES GENES & DEVELOPMENT Coetzee, T., Herschlag, D., Belfort, M. 1994; 8 (13): 1575-1588

    Abstract

    To address the effect of host proteins on the self-splicing properties of the group I introns of bacteriophage T4, we have purified an activity from Escherichia coli extracts that facilitates both trans- and cis-splicing of the T4 introns in vitro. The activity is attributable to a number of proteins, several of which are ribosomal proteins. Although these proteins have variable abilities to stimulate splicing, ribosomal protein S12 is the most effective. The activity mitigates the negative effects on splicing of the large internal open reading frames (ORFs) common to the T4 introns. In contrast to proteins shown previously to facilitate group I splicing, S12 does not bind strongly or specifically to the intron. Rather, S12 binds RNA with broad specificity and can also facilitate the action of a hammerhead ribozyme. Addition of S12 to unreactive trans-splicing precursors promoted splicing, suggesting that S12 can resolve misfolded RNAs. Furthermore, incubation with S12 followed by its proteolytic removal prior to the initiation of the splicing reaction still resulted in splicing enhancement. These results suggest that this protein facilitates splicing by acting as an RNA chaperone, promoting the assembly of the catalytically active tertiary structure of ribozymes.

    View details for Web of Science ID A1994NX54500008

    View details for PubMedID 7958841

  • AN RNA CHAPERONE ACTIVITY OF NONSPECIFIC RNA-BINDING PROTEINS IN HAMMERHEAD RIBOZYME CATALYSIS EMBO JOURNAL Herschlag, D., Khosla, M., Tsuchihashi, Z., Karpel, R. L. 1994; 13 (12): 2913-2924

    Abstract

    We have previously shown that a protein derived from the p7 nucleocapsid (NC) protein of HIV type-1 increases kcat/Km and kcat for cleavage of a cognate substrate by a hammerhead ribozyme. Here we show directly that the increase in kcat/Km arises from catalysis of the annealing of the RNA substrate to the ribozyme and the increase in kcat arises from catalysis of dissociation of the RNA products from the ribozyme. A peptide polymer derived from the consensus sequence of the C-terminal domain of the hnRNP A1 protein (A1 CTD) provides similar enhancements. Although these effects apparently arise from non-specific interactions, not all non-specific binding interactions led to these enhancements. NC and A1 CTD exert their effects by accelerating attainment of the thermodynamically most stable species throughout the ribozyme catalytic cycle. In addition, NC protein is shown to resolve a misfolded ribozyme-RNA complex that is otherwise long lived. These in vitro results suggest that non-specific RNA binding proteins such as NC and hnRNP proteins may have a biological role as RNA chaperones that prevent misfolding of RNAs and resolve RNAs that have misfolded, thereby ensuring that RNA is accessible for its biological functions.

    View details for Web of Science ID A1994NT76100020

    View details for PubMedID 8026476

  • COMPARISON OF PH DEPENDENCIES OF THE TETRAHYMENA RIBOZYME REACTIONS WITH RNA 2'-SUBSTITUTED AND PHOSPHOROTHIOATE SUBSTRATES REVEALS A RATE-LIMITING CONFORMATIONAL STEP BIOCHEMISTRY Herschlag, D., Khosla, M. 1994; 33 (17): 5291-5297

    Abstract

    The L-21 ScaI ribozyme (E) derived from the self-splicing group I intron of Tetrahymena pre-rRNA catalyzes an RNA endonuclease reaction analogous to the first step in self-splicing: CCCUCUAAAAA (S) + G-->CCCUCU+GAAAAA. We show herein that the pH dependence for the single-turnover reaction E.S+G-->products follows a pH dependence with pKapp = 6.9 (10 mM MgCl2, 50 degrees C). This result was surprising because the titratable groups of RNA have pKa values of < approximately 4 or > approximately 9. Thus, two models were considered: (i) the ribozyme structure perturbs a pKa such that the pKapp of 6.9 corresponds to an actual titration or (ii) the pKapp is a kinetic pKa, reflecting a change in the rate-limiting step rather than an actual titration. Oligonucleotide substrates with -H (deoxyribose), -F (2'-fluoro-2'-deoxyribose), and -OH (ribose) substitutions at the 2' position of the U residue at the cleavage site [U(-1)] vary considerably in their intrinsic reactivities. In the ribozyme reaction these substrates reacted at very different rates at low pH, but approached the same limiting reaction rate at high pH. Similarly, substitution of the pro-RP nonbridging oxygen atom of the reactive phosphoryl group by sulfur lowers the intrinsic reactivity of the oligonucleotide substrate. In the ribozyme reaction, this "thio effect" was 2.3 below pH 6.9, whereas the thio substitution had no effect on the rate above pH 6.9.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1994NJ85900036

    View details for PubMedID 8172903

  • A KINETIC AND THERMODYNAMIC FRAMEWORK FOR THE HAMMERHEAD RIBOZYME REACTION BIOCHEMISTRY Hertel, K. J., Herschlag, D., Uhlenbeck, O. C. 1994; 33 (11): 3374-3385

    Abstract

    A hammerhead ribozyme (HH16) with eight potential base pairs in each of the substrate recognition helices stabilized product binding sufficiently to enable investigation of the ligation of oligonucleotides bound to the ribozyme. All individual rate constants for product association and dissociation were determined. The following conclusions were obtained for HH16 from the analysis performed at 50 mM Tris, pH 7.5, 10 mM MgCl2, and 25 degrees C. (1) HH16 cleaves bound substrate with a rate constant of k2 = 1 min-1, similar to rate constants obtained with other hammerhead ribozymes. (2) k-2, the rate of ligation of the 5' product and 3' product to form substrate, equaled 0.008 min-1, indicating an approximately 100-fold preference for the formation of products on the ribozyme. This internal equilibrium, compared with that for the overall solution reaction, gives an effective concentration (EC) of 10(-2) M for the two products bound to the ribozyme. This low EC suggests that upon cleavage of S the hammerhead complex acquires a "floppiness" which provides an entropic advantage for the formation of products on the ribozyme. (3) Product and substrate association rate constants were in the range of 10(7)-10(8) M-1 min-1, comparable to values determined for short helices. (4) The stabilities of ribozyme/product complexes were similar to affinities predicted from helix-coil transitions of simple RNA duplexes, providing no indication of additional tertiary interactions. The products, P1 and P2, stabilize one another 4-fold on the ribozyme. (5) The dissociation constant for the binding of the substrate to the ribozyme was estimated to be about 10(-17) M. These results allowed the construction of a free energy profile for the reaction of HH16, and provide a basis for future mechanistic studies.

    View details for Web of Science ID A1994NC04100031

    View details for PubMedID 8136375

  • PROTEIN ENHANCEMENT OF HAMMERHEAD RIBOZYME CATALYSIS SCIENCE Tsuchihashi, Z., Khosla, M., Herschlag, D. 1993; 262 (5130): 99-102

    Abstract

    When the recognition sequence of a ribozyme is extended beyond a certain length, turnover is slowed and specificity is decreased. Here, it is shown that a protein can help a ribozyme overcome these general limitations on ribozyme activity. Cleavage of an RNA oligonucleotide by a hammerhead ribozyme is enhanced 10- to 20-fold upon addition of a protein derived from the p7 nucleocapsid (NC) protein of human immunodeficiency virus-type 1. The NC protein also enhances the ability of the ribozyme to discriminate between cleavage of RNA oligonucleotides with differing sequences. These catalytic improvements can be attributed to the strand exchange activity of this RNA binding protein. It is conceivable that endogenous or added proteins may provide analogous increases in ribozyme activity and specificity in vivo.

    View details for Web of Science ID A1993LZ63500031

    View details for PubMedID 7692597

  • GUANOSINE BINDING TO THE TETRAHYMENA RIBOZYME - THERMODYNAMIC COUPLING WITH OLIGONUCLEOTIDE BINDING PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA McConnell, T. S., Cech, T. R., Herschlag, D. 1993; 90 (18): 8362-8366

    Abstract

    The L-21 Sca I ribozyme derived from the group I intron of Tetrahymena thermophila pre-rRNA catalyzes an endonuclease reaction analogous to the first step of self-splicing. Guanosine (G) is bound by the ribozyme, and its 3'-hydroxyl group acts as the nucleophile. Here, we provide evidence that Km for G in several single-turnover reactions is equal to the equilibrium dissociation constant for G. This evidence includes the observation that removal of the 2'-hydroxyl group at the cleavage site of the oligoribonucleotide substrate [from CCCUCUA to CCCUC(dU)A] decreases the rate of cleavage approximately 1000-fold but has no effect on either the Km for G (0.17 mM) or for guanosine 5'-monophosphate (pG) (0.09 mM). In the course of this study, it was observed that Km for G or pG was lower by a factor of 5 for reactions with the ribozyme-CCCUC(dU)A complex compared with the free ribozyme, indicating a modest amount of thermodynamic coupled binding of the two substrates. The decrease in the rate of oligonucleotide dissociation upon addition of saturating pG provides independent support for this coupling. Coupling is lost with a substrate that cannot make the normal tertiary interactions with the ribozyme, providing evidence that coupled binding requires docking of the substrate into the catalytic core. Surprisingly, the binding of product CCCUCU and G is slightly anticooperative, indicating that the cleaved pA is important for coupling with substrate. Coupled binding suggests a splicing model in which the intron binds G tightly to promote the first step of reaction, after which its binding is an order of magnitude weaker, thereby facilitating the second step.

    View details for Web of Science ID A1993LX75000014

    View details for PubMedID 8378306

  • CATALYSIS OF THE HYDROLYSIS OF PHOSPHORYLATED PYRIDINES BY ALKALINE-PHOSPHATASE HAS LITTLE OR NO DEPENDENCE ON THE PK(A) OF THE LEAVING GROUP BIOCHEMISTRY Labow, B. I., Herschlag, D., JENCKS, W. P. 1993; 32 (34): 8737-8741

    Abstract

    Bacterial alkaline phosphatase is an active catalyst for the hydrolysis of N-phosphorylated pyridines, with values of the second-order rate constant kcat/Km in the range 0.4-1.2 x 10(6) M-1 s-1 at pH 8.0, 25 degrees C. There is little or no dependence of the rate on the pKa of the leaving group; the value of beta 1g is 0 +/- 0.05, which may be compared with beta 1g = -1.0 for the nonenzymic reaction. Phosphorylated pyridines do not have a free electron pair available for protonation or coordination of the leaving group. Therefore, this result means that the similar, small dependence on leaving group structure for the enzyme-catalyzed hydrolysis of phosphate esters [Hall, A. D., & Williams, A. (1986) Biochemistry 25, 4784-4790) does not provide evidence for general acid catalysis or electrophilic assistance of leaving group expulsion. The results are consistent with the hypothesis that productive binding of the substrate, which may involve a conformational change, is largely rate limiting for turnover of the enzyme at low substrate concentrations.

    View details for Web of Science ID A1993LV97100003

    View details for PubMedID 8395879

  • THE IMPORTANCE OF BEING RIBOSE AT THE CLEAVAGE SITE IN THE TETRAHYMENA RIBOZYME REACTION BIOCHEMISTRY Herschlag, D., Eckstein, F., Cech, T. R. 1993; 32 (32): 8312-8321

    Abstract

    The ribozyme derived from the intron of Tetrahymena thermophila pre-rRNA catalyzes a site-specific endonuclease reaction with both RNA and DNA oligonucleotides. The total transition-state stabilization by the ribozyme, encompassing the binding and chemical steps, is 4.8 kcal/mol greater with a single ribose at the cleavage site relative to the all-deoxyribose substrate. Here we show that this effect is specific to the chemical transition state, with a contribution of only approximately 0.7 kcal/mol toward binding. Substrates with a series of 2'-substituents, -OH(ribo), -F2 (2',2'-difluoro-2'-deoxyribo), F(2'-fluoro-2'-deoxyribo), and -H(deoxyribo), follow a linear free energy relationship between the rate of the chemical step of the ribozyme-catalyzed reaction and the pK(a) of the leaving group, with slope beta leaving group approximately -0.8. Because proton donation to the 3'-oxygen atom from a general acid of the ribozyme would be expected to render the rate insensitive to the pK(a) of the leaving group, it is suggested that this ribozyme does not employ general acid catalysis. The 2'-OCH3 (2'-methoxy-2'-deoxyribo) substituent does not follow this correlation, apparently due to steric hindrance within the active site. The rate of cleavage of the 2'-substituted substrates by the ribozyme follows the order 2'-F2 > -F > -H, suggestive of an inductive effect, i.e., acceleration of the reaction by electron-withdrawing groups. The 2'-OH group provides the largest transition-state stabilization. Because of uncertainty in the relative effect of the 2'-OH and 2'-H substituents on the pK(a) of the neighboring 3'-oxygen leaving group, we do not discount the possibility of interactions between the 2'-hydroxyl group and the ribozyme that further enhance reactivity. Nevertheless, the 2'-OH effect can be explained at least partially by an intramolecular hydrogen bond to an incipient oxyanion at the neighboring 3'-position. This oxyanion is forming as the phosphodiester bond is breaking, explaining why the stabilization is specific to the transition state. Analogous differential hydrogen bonding might be widely used by enzymes to achieve selective transition-state stabilization.

    View details for Web of Science ID A1993LV96900035

    View details for PubMedID 7688573

  • CONTRIBUTIONS OF 2'-HYDROXYL GROUPS OF THE RNA SUBSTRATE TO BINDING AND CATALYSIS BY THE TETRAHYMENA RIBOZYME - AN ENERGETIC PICTURE OF AN ACTIVE-SITE COMPOSED OF RNA BIOCHEMISTRY Herschlag, D., Eckstein, F., Cech, T. R. 1993; 32 (32): 8299-8311

    Abstract

    The ribozyme derived from the intervening sequence of Tetrahymena thermophila pre-rRNA catalyzes a site-specific endonuclease reaction with both RNA and DNA oligonucleotides: CCCUCUAAAAA + G<-->CCCUCU + GAAAAA. However, the RNA substrate (rS) binds approximately 10(4)-fold stronger than the DNA substrate (dS) and once bound reacts approximately 10(4)-fold faster. Here we have investigated the role of individual 2'-hydroxyl groups by comparing the binding and reactivity of "chimeric" oligonucleotide substrates, in which the 2'-substituents of the individual sugar residues have been varied. Chimeric substrates containing a single ribonucleotide at positions -6 to +3 (numbered from the cleavage site) were cleaved faster than dS by factors of 3.5, 3.5, 2.3, 65, 18, 1700, 7.8, 1.7, and 1.4 [(kcat/Km)chimeric S/(kcat/Km)dS]. The sum of the energetic contributions from the individual 2'-hydroxyl groups of 13.3 kcal/mol accounts for the 12.2 kcal/mol greater stabilization for RNA than for DNA in binding and cleavage (i.e., overall transition-state stabilization). This observation and the significant energetic effects from single ribose substitutions at opositions-3 to +1 strongly suggest that local interactions, rather than overall helical differences, largely account for the different binding and reactivity of the DNA and RNA substrates. Each 2'-hydroxyl group was evaluated for its effect on each of three reaction steps leading to the chemical transition state: two binding steps (duplex formation and docking into tertiary interactions) and the chemical cleavage step. The 2'-hydroxyl groups at positions -3 and -2 stabilize docking, and this stabilization is maintained in the chemical step. This "uniform binding" indicates that these interactions contribute to catalysis by positioning the oligonucleotide substrate for reaction. The 2'-hydroxyl at position +1 has a small effect on the binding step and an additional small but significant effect on the chemical step. Thus, the ribozyme, like protein enzymes, can take advantage of interactions away from the site of chemistry to provide stabilization specifically in the transition state. The 2'-hydroxyl at position -1 exerts its large effect nearly exclusively on the chemical step [Herschlag, D., Eckstein, F., & Cech, T.R. (1993) Biochemistry (following paper in this issue)]. The energetic effects of other modifications of the 2'-substituents provide a crude picture of the active site. The 2'-OCH3 substituent at position -3 inhibits the reaction approximately 10-fold relative to 2'-H, suggesting than an unfavorable interaction cannot be avoided by an isoenergetic structural rearrangement.(ABSTRACT TRUNCATED AT 400 WORDS)

    View details for Web of Science ID A1993LV96900034

    View details for PubMedID 7688572

  • SYNERGISM IN TRANSCRIPTIONAL ACTIVATION - A KINETIC VIEW GENES & DEVELOPMENT Herschlag, D., Johnson, F. B. 1993; 7 (2): 173-179

    View details for Web of Science ID A1993KM85900001

    View details for PubMedID 8436289

  • MUTATIONS AT THE GUANOSINE-BINDING SITE OF THE TETRAHYMENA RIBOZYME ALSO AFFECT SITE-SPECIFIC HYDROLYSIS NUCLEIC ACIDS RESEARCH Legault, P., Herschlag, D., Celander, D. W., Cech, T. R. 1992; 20 (24): 6613-6619

    Abstract

    Self-splicing group I introns use guanosine as a nucleophile to cleave the 5' splice site. The guanosine-binding site has been localized to the G264-C311 base pair of the Tetrahymena intron on the basis of analysis of mutations that change the specificity of the nucleophile from G (guanosine) to 2AP (2-aminopurine ribonucleoside) (F. Michel et al. (1989) Nature 342, 391-395). We studied the effect of these mutations (G-U, A-C and A-U replacing G264-C311) in the L-21 ScaI version of the Tetrahymena ribozyme. In this enzymatic system (kcat/Km)G monitors the cleavage step. This kinetic parameter decreased by at least 5 x 10(3) when the G264-C311 base pair was mutated to an A-U pair, while (kcat/Km)2AP increased at least 40-fold. This amounted to an overall switch in specificity of at least 2 x 10(5). The nucleophile specificity (G > 2AP for the G-C and G-U pairs, 2AP > G for the A-U and A-C pairs) was consistent with the proposed hydrogen bond between the nucleotide at position 264 and N1 of the nucleophile. Unexpectedly, the A-U and A-C mutants showed a decrease of an order of magnitude in the rate of ribozyme-catalyzed hydrolysis of RNA, in which H2O or OH- replaces G as the nucleophile, whereas the G-U mutant showed a decrease of only 2-fold. The low hydrolysis rates were not restored by raising the Mg2+ concentration or lowering the temperature. In addition, the mutant ribozymes exhibited a pattern of cleavage by Fe(II)-EDTA indistinguishable from that of the wild type, and the [Mg2+]1/2 for folding of the A-U mutant ribozyme was the same as that of the wild type. Therefore the guanosine-binding site mutations do not appear to have a major effect on RNA folding or stability. Because changing G264 affects the hydrolysis reaction without perturbing the global folding of the RNA, we conclude that the catalytic role of this conserved nucleotide is not limited to guanosine binding.

    View details for Web of Science ID A1992KG70500024

    View details for PubMedID 1480482

  • RNA CATALYSIS BY A GROUP-I RIBOZYME - DEVELOPING A MODEL FOR TRANSITION-STATE STABILIZATION JOURNAL OF BIOLOGICAL CHEMISTRY Cech, T. R., Herschlag, D., Piccirilli, J. A., Pyle, A. M. 1992; 267 (25): 17479-17482

    View details for Web of Science ID A1992JM22300001

    View details for PubMedID 1381347

  • EVIDENCE FOR PROCESSIVITY AND 2-STEP BINDING OF THE RNA SUBSTRATE FROM STUDIES OF J1/2 MUTANTS OF THE TETRAHYMENA RIBOZYME BIOCHEMISTRY Herschlag, D. 1992; 31 (5): 1386-1399

    Abstract

    J1/2 of the Tetrahymena ribozyme, a sequence of three A residues, connects the RNA-binding site to the catalytic core. Addition or deletion of bases from J1/2 improves turnover and substrate specificity in the site-specific endonuclease reaction catalyzed by this ribozyme: G2CCCUCUA5 (S) + G in-equilibrium G2CCCUCU (P) + GA5. These paradoxical enhancements are caused by decreased affinity of the ribozyme for S and P [Young, B., Herschlag, D., & Cech, T.R. (1991) Cell 67, 1007]. An additional property of these mutant ribozymes, decreased fidelity of RNA cleavage, is now analyzed. (Fidelity is the ability to cleave at the correct phosphodiester bond within a particular RNA substrate.) Introduction of deoxy residues to give "chimeric" ribo/deoxyribooligonucleotides changes the positions of incorrect cleavage. Previous work indicated that S is bound to the ribozyme by both base pairing and teritary interactions involving 2'-hydroxyl groups of S. The data herein strongly suggest that the P1 duplex, which consists of S base-paired with the 5' exon binding site of the ribozyme, can dock into tertiary interactions in different registers; different 2'-hydroxyl groups of S plug into tertiary contacts with the ribozyme in the different registers. It is concluded that the mutations decrease fidelity by increasing the probability of docking out of register relative to docking in the normal register, thereby giving cleavage at different positions along S. These data also show that the contribution of J1/2 to the teritiary interactions is indirect, not direct. Thus, a structural role of the nonconserved J1/2 is indicated: this sequence positions S to optimize tertiary binding interactions and to ensure cleavage at the phosphodiester bond corresponding to the 5' splice site. Substitution of sulfur for the nonbridging pro-RP oxygen atom at the normal cleavage site has no effect on (kcat/Km)S but decreases the fraction of cleavage at the normal site in reactions catalyzed by the -3A mutant ribozyme, which has all three A residues of J1/2 removed. Thus, the ribozyme chooses where to cleave S after rate-limiting binding of S, indicating that docking can change after binding and suggesting that the ribozyme could act processively. Indeed, it is shown that the +2A ribozyme cleaves at one position along an RNA substrate and then, before releasing that RNA product, cleaves it again.(ABSTRACT TRUNCATED AT 400 WORDS)

    View details for Web of Science ID A1992HD15700015

    View details for PubMedID 1736996

  • MUTATIONS IN A NONCONSERVED SEQUENCE OF THE TETRAHYMENA RIBOZYME INCREASE ACTIVITY AND SPECIFICITY CELL Young, B., Herschlag, D., Cech, T. R. 1991; 67 (5): 1007-1019

    Abstract

    The RNA substrate-binding site of the Tetrahymena ribozyme is connected to the catalytic core by the joining region J1/2. Although J1/2 is not conserved among group I introns, small insertions or deletions in this sequence have dramatic effects, enhancing the turnover number and sequence specificity of ribozyme-catalyzed RNA cleavage. Measurements of rate constants for individual steps in the reaction have revealed the basis of these improvements. Ironically, the higher turnover and specificity both result from decreased affinity for RNA, rather than better cleavage. These results provide evidence that the nonconserved J1/2 sequence positions the RNA substrate to optimize tertiary interactions and ensure cleavage at the position corresponding to the 5' splice site. The wild-type RNA is well adapted to its biological function, and its limitations in multiple turnover can be corrected by mutation.

    View details for Web of Science ID A1991GT75500019

    View details for PubMedID 1959129

  • IMPLICATIONS OF RIBOZYME KINETICS FOR TARGETING THE CLEAVAGE OF SPECIFIC RNA MOLECULES INVIVO - MORE ISNT ALWAYS BETTER PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Herschlag, D. 1991; 88 (16): 6921-6925

    Abstract

    Kinetic and thermodynamic factors that determine specificity of RNA cleavage by ribozymes are illustrated with examples from recent work with a ribozyme derived from the group I intron of Tetrahymena thermophila pre-rRNA. The conclusions also apply to other ribozymes, to antisense oligonucleotide experiments, and to RNA and DNA cleavage agents that can recognize a single-stranded or double-stranded region of variable length. At first, adding bases to a ribozyme's recognition sequence is expected to increase cleavage of the target RNA relative to cleavage of other RNAs. However, adding more bases ultimately reduces this discrimination, as cleavage occurs essentially every time the target RNA or a mismatched RNA binds the ribozyme. This occurs despite the weaker binding of the mismatched RNA because dissociation becomes too slow (binding is too strong) to allow the ribozyme to "choose" between cleavage of the target RNA and a mismatched RNA. In summary, more (base pairing) isn't always better, because maximal discrimination requires equilibrium binding prior to cleavage. The maximum discrimination that can be obtained is expected to be greater with an A + U-rich recognition sequence than with a G + C-rich recognition sequence. This is because the weaker A.U base pairs (relative to G-C base pairs) allow recognition to be spread over a larger number of bases while preventing binding that is too strong. Finally, creating an A-rich ribozyme rather than a U-rich ribozyme avoids the loss in discrimination expected with U-rich ribozymes from the formation of U.G wobble pairs in addition to the "targeted" Watson-Crick U.A pair.

    View details for Web of Science ID A1991GB70100005

    View details for PubMedID 1871108

  • RIBOZYME-CATALYZED AND NONENZYMATIC REACTIONS OF PHOSPHATE DIESTERS - RATE EFFECTS UPON SUBSTITUTION OF SULFUR FOR A NONBRIDGING PHOSPHORYL OXYGEN ATOM BIOCHEMISTRY Herschlag, D., Piccirilli, J. A., Cech, T. R. 1991; 30 (20): 4844-4854

    Abstract

    The L-21 ScaI ribozyme derived from the intervening sequence of Tetrahymena thermophila pre-rRNA catalyzes a guanosine-dependent endonuclease reaction that is analogous to the first step in self-splicing of this intervening sequence. We now describe pre-steady-state kinetic experiments, with sulfur substituting for the pro-RP (nonbridging) phosphoryl oxygen atom at the site of cleavage, that test aspects of a kinetic model proposed for the ribozyme reaction (Herschlag, D., & Cech, T. R. (1990) Biochemistry 29, 10159-10171). Thio substitution does not affect the reaction with subsaturating oligonucleotide substrate and saturating guanosine ((kcat/Km)S), consistent with the previous finding that binding of the oligonucleotide substrate limits this rate constant. In contrast, there is a significant decrease in the rate of single-turnover reactions of ribozyme-bound (i.e., saturating) oligonucleotide substrate upon thio substitution, with decreases of 2.3-fold for the reaction with guanosine ((kcat/Km)G) and 7-fold for hydrolysis [i.e., with solvent replacing guanosine; kc(-G)]. These "thio effects" are consistent with rate-limiting chemistry, as shown by comparison with model reactions. Nonenzymatic nucleophilic substitution reactions of the phosphate diester, methyl 2,4-dinitrophenyl phosphate monoanion, are slowed 4-11-fold by thio substitution for reactions with hydroxide ion, formate ion, fluoride ion, pyridine, and nicotinamide. In addition, we have confirmed that thio substitution has no effect on the nonenzymatic alkaline cleavage of RNA (Burgers, P. M. J., & Eckstein, F. (1979) Biochemistry 18, 592-596). Considering the strong preference of Mg2+ for binding to oxygen rather than sulfur, the modest thio effect on the chemical step of the ribozyme-catalyzed reaction and the absence of a thio effect on the equilibrium constant for binding of the oligonucleotide substrate suggest that the pro-RP oxygen atom is not coordinated to Mg2+ in the E.S complex or in the transition state. General implications of thio effects in enzymatic reactions of phosphate diesters are discussed.

    View details for Web of Science ID A1991FM71200003

    View details for PubMedID 2036355

  • CATALYSIS OF RNA CLEAVAGE BY THE TETRAHYMENA-THERMOPHILA RIBOZYME .1. KINETIC DESCRIPTION OF THE REACTION OF AN RNA SUBSTRATE COMPLEMENTARY TO THE ACTIVE-SITE BIOCHEMISTRY Herschlag, D., Cech, T. R. 1990; 29 (44): 10159-10171

    Abstract

    A ribozyme derived from the intervening sequence (IVS) of the Tetrahymena preribosomal RNA catalyzes a site-specific endonuclease reaction: G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine). This reaction is analogous to the first step in self-splicing of the pre-rRNA, with the product G2CCCUCU analogous to the 5'-exon. The following mechanistic conclusions have been derived from pre-steady-state and steady-state kinetic measurements at 50 degrees C and neutral pH in the presence of 10 mM Mg2+. The value of kcat/Km = 9 x 10(7) M-1 min-1 for the oligonucleotide substrate with saturating G represents rate-limiting binding. This rate constant for binding is of the order expected for formation of a RNA.RNA duplex between oligonucleotides. (Phylogenetic and mutational analyses have shown that this substrate is recognized by base pairing to a complementary sequence within the IVS). The value of kcat = 0.1 min-1 represents rate-limiting dissociation of the 5'-exon analogue, G2CCCUCU. The product GA5 dissociates first from the ribozyme because of this slow off-rate for G2CCCUCU. The similar binding of the product, G2CCCUCU, and the substrate, G2CCCUCUA5, to the 5'-exon binding site of the ribozyme, with Kd = 1-2 nM, shows that the pA5 portion of the substrate makes no net contribution to binding. Both the substrate and product bind approximately 10(4)-fold (6 kcal/mol) stronger than expected from base pairing with the 5'-exon binding site. Thus, tertiary interactions are involved in binding. Binding of G2CCCUCU and binding of G are independent. These and other data suggest that binding of the oligonucleotide substrate, G2CCCUCUA5, and binding of G are essentially random and independent. The rate constant for reaction of the ternary complex is calculated to be kc approximately equal to 350 min-1, a rate constant that is not reflected in the steady-state rate parameters with saturating G. The simplest interpretation is adopted, in which kc represents the rate of the chemical step. A site-specific endonuclease reaction catalyzed by the Tetrahymena ribozyme in the absence of G was observed; the rate of the chemical step with solvent replacing guanosine, kc(-G) = 0.7 min-1, is approximately 500-fold slower than that with saturating guanosine. The value of kcat/Km = 6 x 10(7) M-1 min-1 for this hydrolysis reaction is only slightly smaller than that with saturating guanosine, because the binding of the oligonucleotide substrate is predominantly rate-limiting in both cases.(ABSTRACT TRUNCATED AT 400 WORDS)

    View details for Web of Science ID A1990EG22200003

    View details for PubMedID 2271645

  • CATALYSIS OF RNA CLEAVAGE BY THE TETRAHYMENA-THERMOPHILA RIBOZYME .2. KINETIC DESCRIPTION OF THE REACTION OF AN RNA SUBSTRATE THAT FORMS A MISMATCH AT THE ACTIVE-SITE BIOCHEMISTRY Herschlag, D., Cech, T. R. 1990; 29 (44): 10172-10180

    Abstract

    The site-specific endonuclease reaction catalyzed by the ribozyme from the Tetrahymena pre-rRNA intervening sequence has been characterized with a substrate that forms a "matched" duplex with the 5' exon binding site of the ribozyme [G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine); Herschlag, D., & Cech, T.R. (1990) Biochemistry (preceding paper in this issue)]. The rate-limiting step with saturating substrate is dissociation of the product G2CCCUCU. Here we show that the reaction of the substrate G2CCCGCUA5, which forms a "mismatched" duplex with the 5' exon binding site at position -3 from the cleavage site, has a value of kcat that is approximately 10(2)-fold greater than kcat for the matched substrate (50 degrees C, 10 mM MgCl2, pH 7). This is explained by the faster dissociation of the mismatched product, G2CCCGCU, than the matched product. With subsaturating oligonucleotide substrate and saturating G, the binding of the oligonucleotide substrate and the chemical step are each partially rate-limiting. The rate constant for the chemical step of the endonuclease reaction and the rate constant for the site-specific hydrolysis reaction, in which solvent replaces G, are each within approximately 2-fold with the matched and mismatched substrates, despite the approximately 10(3)-fold weaker binding of the mismatched substrate. This can be described as "uniform binding" of the base at position -3 in the ground state and transition state [Albery, W.J., & Knowles, J. R. (1976) Biochemistry 15, 5631-5640]. Thus, the matched substrate does not use its extra binding energy to preferentially stabilize the transition state.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1990EG22200004

    View details for PubMedID 2271646

  • CATALYSIS OF THE HYDROLYSIS OF PHOSPHORYLATED PYRIDINES BY MG(OH)+ - A POSSIBLE MODEL FOR ENZYMATIC PHOSPHORYL TRANSFER BIOCHEMISTRY Herschlag, D., JENCKS, W. P. 1990; 29 (21): 5172-5179

    Abstract

    The second-order rate constants for reaction of the Mg2+ complexes of phosphorylated pyridine monoanions with Mg(OH)+ are 10(4)-10(6)-fold larger than the second-order rate constants for their reaction with water (25 degrees C, ionic strength 1.5). Of the 10(6)-fold rate enhancement with the phosphorylated 4-morpholinopyridine/Mg2 complex, approximately 10(4)-fold is attributed to the greater nucleophilicity of Mg(OH)+ compared with water. The remaining catalysis of approximately 10(2)-fold is attributed to induced intramolecularity from positioning of the hydroxide ion and phosphoryl group by the Mg2+ ions. This reaction may provide a model for the role of a metal ion in increasing the concentration of the anions of enolpyruvate and serine and holding the nucleophile in the correct position for phosphoryl transfer in the reactions catalyzed by pyruvate kinase and alkaline phosphatase, for example. Some mechanisms that can provide catalysis of phosphoryl transfer through a metaphosphate-like transition state are reviewed briefly.

    View details for Web of Science ID A1990DF84300025

    View details for PubMedID 2378873

  • DNA CLEAVAGE CATALYZED BY THE RIBOZYME FROM TETRAHYMENA NATURE Herschlag, D., Cech, T. R. 1990; 344 (6265): 405-409

    Abstract

    An RNA enzyme derived from the self-splicing intervening sequence of Tetrahymena thermophila catalyses sequence-specific cleavage of an oligodeoxyribonucleotide substrate. Compared with RNA, the DNA substrate is bound very weakly and is cleaved very slowly, revealing the importance of the RNA 2'-hydroxyl group in both the binding and chemical steps. The finding that catalysis by RNA can extend to DNA substrates indicates new possibilities for the transposition of intervening sequences and for the design of DNA cleavage agents with novel sequence specificities.

    View details for Web of Science ID A1990CW47800054

    View details for PubMedID 1690858

  • NUCLEOPHILES OF HIGH REACTIVITY IN PHOSPHORYL TRANSFER-REACTIONS - ALPHA-EFFECT COMPOUNDS AND FLUORIDE-ION JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Herschlag, D., JENCKS, W. P. 1990; 112 (5): 1951-1956
  • THE EFFECTS OF MG-2+, HYDROGEN-BONDING, AND STERIC FACTORS ON RATE AND EQUILIBRIUM-CONSTANTS FOR PHOSPHORYL TRANSFER BETWEEN CARBOXYLATE IONS AND PYRIDINES JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Herschlag, D., JENCKS, W. P. 1990; 112 (5): 1942-1950
  • Nucleophiles of High Reactivity in Phosphoryl Transfer Reactions: &#945;-Effect Compounds and Fluoride Ion. J. Am. Chem. Soc. Herschlag, D., Jencks, W.P. 1990; 112: 1951-1956
  • The Effect of Mg2+, Hydrogen Bonding and Steric Factors on Rate and Equilibrium Constants for Phosphoryl Transfer between Carboxylate Ions and Pyridines. J. Am. Chem. Soc. Herschlag, D., Jencks, W.P. 1990; 112: 1942-1950
  • PHOSPHORYL TRANSFER TO ANIONIC OXYGEN NUCLEOPHILES - NATURE OF THE TRANSITION-STATE AND ELECTROSTATIC REPULSION JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Herschlag, D., JENCKS, W. P. 1989; 111 (19): 7587-7596
  • EVIDENCE THAT META-PHOSPHATE MONOANION IS NOT AN INTERMEDIATE IN SOLVOLYSIS REACTIONS IN AQUEOUS-SOLUTION JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Herschlag, D., JENCKS, W. P. 1989; 111 (19): 7579-7586
  • Phosphoryl Transfer to Oxyanions: The Nature of the Transition State and Electrostatic Repulsion. J. Am. Chem. Soc. Herschlag, D., Jencks, W.P. 1989; 111: 7587-7596
  • Evidence That Metaphosphate is Not an Intermediate in Solvolysis Reactions in Aqueous Solution J. Am. Chem. Soc. Herschlag, D., Jencks, W.P. 1989; 111: 7579-7586
  • The Role of Induced Fit and Conformational Changes of Enzymes in Specificity and Catalysis. Bioorganic Chemistry Herschlag, D. 1988; 16: 62-96
  • THE EFFECT OF DIVALENT METAL-IONS ON THE RATE AND TRANSITION-STATE STRUCTURE OF PHOSPHORYL-TRANSFER REACTIONS JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Herschlag, D., JENCKS, W. P. 1987; 109 (15): 4665-4674
  • The Effect of Divalent Metal Ions on the Rate and Transition State Structure of Phosphoryl Transfer Reactions. J. Am. Chem. Soc. Herschlag, D., Jencks, W.P. 1987; 109: 4665-4674
  • PYROPHOSPHATE FORMATION FROM ACETYL PHOSPHATE AND ORTHO-PHOSPHATE ANIONS IN CONCENTRATED AQUEOUS SALT-SOLUTIONS DOES NOT PROVIDE EVIDENCE FOR A META-PHOSPHATE INTERMEDIATE JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Herschlag, D., JENCKS, W. P. 1986; 108 (25): 7938-7946
  • Pyrophosphate Formation from Acetyl Phosphate and Orthophosphate Anions in Concentrated Aqueous Salt Solutions Does Not Provide Evidence for a Metaphosphate Intermediate J. Am. Chem. Soc. Herschlag, D., Jencks, W.P. 1986; 108: 7938
  • Decreasing Reactivity with Increasing Nucleophile Basicity. The Effect of Solvation on &#946;nuc for Phosphoryl Transfer to Amines J. Am. Chem. Soc. Jencks, W.P., Haber, M.T., Herschlag, D., Nazaretian, K.L. 1986; 108: 479
  • GALACTOFURANOSYL-CONTAINING GLYCOPEPTIDE OF PENICILLIUM-CHARLESII - VACUUM ULTRAVIOLET CIRCULAR-DICHROISM INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH Herschlag, D., Stevens, E. S., Gander, J. E. 1983; 22 (1): 16-20

    Abstract

    The far and vacuum u.v. circular dichroism (CD) of peptidophosphogalactomannan from P. charlesii is reported to 182.5 nm in aqueous and aqueous/organic solvents, and to 150nm in films. CD of films of the peptide-free derivative is reported to 150 nm. On the basis of these data we conclude that the peptide chain is unordered, and may best be described as a hydrated coil showing some stiffness. The small observed saccharide CD may result from cancellation of contributions from the various saccharide structures present or from a lack of repeating secondary structure.

    View details for Web of Science ID A1983QX64600003

    View details for PubMedID 6224750

Conference Proceedings


  • Nucleic Acid Conformation Ensembles Revealed by Au-SAXS Interferometry Shi, X., Harbury, P., Herschlag, D. CELL PRESS. 2013: 502A-502A
  • RNA Structure, Function, and (Thermo-) Dynamics: A SAXS and Single-Molecule Perspective Lipfert, J., Herschlag, D., Doniach, S., Dekker, N. H. CELL PRESS. 2011: 1-2
  • How enzymes work Herschlag, D. FEDERATION AMER SOC EXP BIOL. 2010
  • Force and Form in RNA Folding Herschlag, D. ADENINE PRESS. 2009: 813-813
  • BIOL 102-Demystifying RNA folding Herschlag, D. AMER CHEMICAL SOC. 2008
  • BIOL 100-Transition state complementarity revisited: Dissecting electrostatic and geometrical catalysis in enzyme active sites Herschlag, D. AMER CHEMICAL SOC. 2008
  • PHYS 212-Traversing the RNA folding landscape Herschlag, D. AMER CHEMICAL SOC. 2007
  • Insights from global studies of eukaryotic translation Herschlag, D. FEDERATION AMER SOC EXP BIOL. 2007: A92-A92
  • Low-resolution models for nucleic acids from small-angle X-ray scattering with applications to electrostatic modeling Lipfert, J., Chu, V. B., Bai, Y., Herschlag, D., Doniach, S. WILEY-BLACKWELL. 2007: S229-S234
  • Traversing the RNA folding landscape Herschlag, D. AMER CHEMICAL SOC. 2007
  • Quantitative and comprehensive decomposition of the ion atmosphere around nucleic acids Bai, Y., Travers, K., Chu, V. B., Lipfert, J., Doniach, S., Herschlag, D. CELL PRESS. 2007: 46A-46A
  • Modeling RNA low resolution structure and thermodynamics from small-angle X-ray scattering Lipfert, J., Chu, V. B., Bai, Y., Ouellet, J., Lilley, D. M., Herschlag, D., Doniach, S. CELL PRESS. 2007: 417A-417A
  • Direct measurement of the full sequence-dependent folding landscape of single nucleic acids using an optical trap Woodside, M. T., Amhony, P. C., Larizadeh, K., Behnke-Parks, W. M., Herschlag, D., Block, S. M. CELL PRESS. 2007: 351A-351A
  • BIOL 181 - Dissecting the fundamental components of enzymatic catalysis: Electrostatic and geometrical catalysis Herschlag, D. AMER CHEMICAL SOC. 2006: 942-942
  • Investigating mechanistic and evolutionary relationships between members of the alkaline phosphatase superfamily Wiersma, H. I., Herschlag, D. FEDERATION AMER SOC EXP BIOL. 2006: A43-A43
  • The structure of nucleotide pyrophosphatase/phosphodiesterase and implications for enzyme evolution in the alkaline phosphatase superfamily Zalatan, J., Fenn, T., Brunger, A., Herschlag, D. FEDERATION AMER SOC EXP BIOL. 2006: A477-A477
  • Dissecting ribozyme mechanisms: Lessons learned about RNA and catalysis Herschlag, D. AMER CHEMICAL SOC. 2005: U500-U500
  • Solving RNA folds with multiplexed radical sources Das, R. J., Herschlag, D. CELL PRESS. 2005: 192A-193A
  • In search of the active site in RNA catalysis. Herschlag, D. AMER CHEMICAL SOC. 2003: U193-U193
  • In search of the active site in RNA catalysis. Herschlag, D. AMER CHEMICAL SOC. 2003: 8652-8652
  • Steady-state and time-resolved properties of dyes bound to the ketosteroid isomerase active site Kraut, D. A., McAnaney, T. B., Boxer, S., Herschlag, D. CELL PRESS. 2003: 169A-169A
  • Exploring the RNA folding landscape Herschlag, D. CELL PRESS. 2003: 308A-308A
  • Rapid electostatic collaspe during RNA folding. Herschlag, D. AMER CHEMICAL SOC. 2002: C88-C88
  • Dissection of an RNA enzyme reveals principles of biological catalysis Herschlag, D. FEDERATION AMER SOC EXP BIOL. 2002: A136-A136
  • Identifying ligands within the Tetrahymena ribozyme that bind and position the catalytic metal ions. Piccirilli, J., Hougland, J. L., Kravchuk, A. V., Herschlag, D. AMER CHEMICAL SOC. 2001: U145-U145
  • Channels in the RNA folding landscape. Herschlag, D. AMER CHEMICAL SOC. 2001: U136-U136
  • Channels in the RNA folding landscape. Herschlag, D. AMER CHEMICAL SOC. 2001: 8635-8635
  • Identifying ligands within the Tetrahymena ribozyme that bind and position the catalytic metal ions. Hougland, J. L., Kravchuk, A. V., Herschlag, D., Piccirilli, J. AMER CHEMICAL SOC. 2001: 8647-8648
  • A single molecule study of the folding dynamics of a large RNA enzyme Babcock, H., Zhuang, X. W., Russell, R., Bartley, L., Herschlag, D., Chu, S. CELL PRESS. 2001: 201A-201A
  • Exploring the RNA folding landscape, one molecule at a time. Herschlag, D., Russell, R., Zhuang, X. W., Bartley, L., Babcock, H. P., Miller, I. S., Doniach, S., Chu, S. AMER CHEMICAL SOC. 2000: U82-U82
  • Enzymatic activity and folding of single RNA molecules Zhuang, X., Bartley, L., Babcock, H., Russell, R., Ha, T., Herschlag, D., Chu, S. FEDERATION AMER SOC EXP BIOL. 2000: A1587-A1587
  • Phosphoryl transfer: Transition states, catalysis and evolution of enzyme diversity Herschlag, D. FEDERATION AMER SOC EXP BIOL. 1999: A1424-A1424
  • Biological catalysis: Lessons from the comparison of RNA and protein enzymes Herschlag, D., Narlikar, G. J., Peracchi, A., Shan, S. FEDERATION AMER SOC EXP BIOL. 1997: A852-A852
  • Phosphoryl transfer to nitrogen nucleophiles: Enzymatic and model studies. Admiraal, S., Herschlag, D. FEDERATION AMER SOC EXP BIOL. 1997: A1317-A1317
  • Catalytic proficiency of E-coli alkaline phosphatase. O'Brien, P., Herschlag, D. FEDERATION AMER SOC EXP BIOL. 1997: A1318-A1318
  • Biological catalysis: From ribozymes to ras. Herschlag, D. AMER CHEMICAL SOC. 1996: 3-BIOL
  • Biological catalysis: From ribozymes to RAS. Herschlag, D. AMER CHEMICAL SOC. 1996: 3-3