Single-Molecule Fluorescence Applied to Translation.
Cold Spring Harbor perspectives in biology
Single-molecule fluorescence methods have illuminated the dynamics of the translational machinery. Structural and bulk biochemical experiments have provided detailed atomic and global mechanistic views of translation, respectively. Single-molecule studies of translation have bridged these views by temporally connecting the conformational and compositional states defined from structural data within the mechanistic framework of translation produced from biochemical studies. Here, we discuss the context for applying different single-molecule fluorescence experiments, and present recent applications to studying prokaryotic and eukaryotic translation. We underscore the power of observing single translating ribosomes to delineate and sort complex mechanistic pathways during initiation and elongation, and discuss future applications of current and improved technologies.
View details for DOI 10.1101/cshperspect.a032714
View details for PubMedID 29891562
Structural Characterization of the HIV-1 Reverse Transcriptase Initiation Complex
CELL PRESS. 2018: 193A
View details for Web of Science ID 000430439600219
Architecture of an HIV-1 reverse transcriptase initiation complex.
Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in viral infection 1 and a common target of antiretroviral drugs 2 . The reaction is catalysed by viral reverse transcriptase (RT)3,4 that is packaged in an infectious virion with two copies of viral genomic RNA 5 each bound to host lysine 3 transfer RNA (tRNALys3), which acts as a primer for initiation of reverse transcription6,7. Upon viral entry into cells, initiation is slow and non-processive compared to elongation8,9. Despite extensive efforts, the structural basis of RT function during initiation has remained a mystery. Here we use cryo-electron microscopy to determine a three-dimensional structure of an HIV-1 RT initiation complex. In our structure, RT is in an inactive polymerase conformation with open fingers and thumb and with the nucleic acid primer-template complex shifted away from the active site. The primer binding site (PBS) helix formed between tRNALys3 and HIV-1 RNA lies in the cleft of RT and is extended by additional pairing interactions. The 5' end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the initiation complex alters RT conformation to decrease activity, highlighting a potential target for drug action.
View details for DOI 10.1038/s41586-018-0055-9
View details for PubMedID 29695867
Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site
2016; 22 (11): 1689-1698
Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5' UTR of the HIV genomic RNA (vRNA) and a host primer tRNA(Lys)3 Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activation signal (PAS) and the primer tRNA occurs in one of these conformers. This intermolecular PAS interaction likely induces strain on a vRNA intramolecular helix, which must be broken for reverse transcription to initiate. We propose a mechanism by which this vRNA/tRNA conformer relieves the kinetic block formed by the vRNA intramolecular helix to initiate reverse transcription.
View details for DOI 10.1261/rna.056804.116
View details for Web of Science ID 000385907600004
View details for PubMedID 27613581
View details for PubMedCentralID PMC5066621
Amino acid sequence repertoire of the bacterial proteome and the occurrence of untranslatable sequences
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (26): 7166-7170
Bioinformatic analysis of Escherichia coli proteomes revealed that all possible amino acid triplet sequences occur at their expected frequencies, with four exceptions. Two of the four underrepresented sequences (URSs) were shown to interfere with translation in vivo and in vitro. Enlarging the URS by a single amino acid resulted in increased translational inhibition. Single-molecule methods revealed stalling of translation at the entrance of the peptide exit tunnel of the ribosome, adjacent to ribosomal nucleotides A2062 and U2585. Interaction with these same ribosomal residues is involved in regulation of translation by longer, naturally occurring protein sequences. The E. coli exit tunnel has evidently evolved to minimize interaction with the exit tunnel and maximize the sequence diversity of the proteome, although allowing some interactions for regulatory purposes. Bioinformatic analysis of the human proteome revealed no underrepresented triplet sequences, possibly reflecting an absence of regulation by interaction with the exit tunnel.
View details for DOI 10.1073/pnas.1606518113
View details for Web of Science ID 000379033400059
View details for PubMedID 27307442
- The molecular choreography of protein synthesis: translational control, regulation, and pathways QUARTERLY REVIEWS OF BIOPHYSICS 2016; 49
- Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II NATURE METHODS 2016; 13 (1): 59-?
The Impact of Aminoglycosides on the Dynamics of Translation Elongation
2013; 3 (2): 497-508
Inferring antibiotic mechanisms on translation through static structures has been challenging, as biological systems are highly dynamic. Dynamic single-molecule methods are also limited to few simultaneously measurable parameters. We have circumvented these limitations with a multifaceted approach to investigate three structurally distinct aminoglycosides that bind to the aminoacyl-transfer RNA site (A site) in the prokaryotic 30S ribosomal subunit: apramycin, paromomycin, and gentamicin. Using several single-molecule fluorescence measurements combined with structural and biochemical techniques, we observed distinct changes to translational dynamics for each aminoglycoside. While all three drugs effectively inhibit translation elongation, their actions are structurally and mechanistically distinct. Apramycin does not displace A1492 and A1493 at the decoding center, as demonstrated by a solution nuclear magnetic resonance structure, causing only limited miscoding; instead, it primarily blocks translocation. Paromomycin and gentamicin, which displace A1492 and A1493, cause significant miscoding, block intersubunit rotation, and inhibit translocation. Our results show the power of combined dynamics, structural, and biochemical approaches to elucidate the complex mechanisms underlying translation and its inhibition.
View details for DOI 10.1016/j.celrep.2013.01.027
View details for Web of Science ID 000321895200022
View details for PubMedID 23416053
RNA purification by preparative polyacrylamide gel electrophoresis.
Methods in enzymology
2013; 530: 315-330
Preparative polyacrylamide gel electrophoresis (PAGE) is a powerful tool for purifying RNA samples. Denaturing PAGE allows separation of nucleic acids that differ by a single nucleotide in length. It is commonly used to separate and purify RNA species after in vitro transcription, to purify naturally occurring RNA variants such as tRNAs, to remove degradation products, and to purify labeled RNA species. To preserve RNA integrity following purification, RNA is usually visualized by UV shadowing or stained with ethidium bromide or SYBR green dyes.
View details for DOI 10.1016/B978-0-12-420037-1.00017-8
View details for PubMedID 24034329
Secondary Structure of the HIV Reverse Transcription Initiation Complex by NMR
JOURNAL OF MOLECULAR BIOLOGY
2011; 410 (5): 863-874
Initiation of reverse transcription of genomic RNA is a key early step in replication of the human immunodeficiency virus (HIV) upon infection of a host cell. Viral reverse transcriptase initiates from a specific RNA-RNA complex formed between a host transfer RNA (tRNA(Lys)(3)) and a region at the 5' end of genomic RNA; the 3' end of the tRNA acts as a primer for reverse transcription of genomic RNA. We report here the secondary structure of the HIV genomic RNA-human tRNA(Lys)(3) initiation complex using heteronuclear nuclear magnetic resonance methods. We show that both RNAs undergo large-scale conformational changes upon complex formation. Formation of the 18-bp primer helix with the 3' end of tRNA(Lys)(3) drives large conformational rearrangements of the tRNA at the 5' end while maintaining the anticodon loop for potential loop-loop interactions. HIV RNA forms an intramolecular helix adjacent to the intermolecular primer helix. This helix, which must be broken by reverse transcription, likely acts as a kinetic block to reverse transcription.
View details for DOI 10.1016/j.jmb.2011.04.024
View details for Web of Science ID 000293674100007
View details for PubMedID 21763492
Probing the conformation of human tRNA(3)(Lys) in solution by NMR
2007; 581 (27): 5307-5314
Human tRNA(3)(Lys) acts as a primer for the reverse transcription of human immunodeficiency virus genomic RNA. To form an initiation complex with genomic RNA, tRNA(3)(Lys) must reorganize its secondary structure. To provide a starting point for mechanistic studies of the formation of the initiation complex, we here present solution NMR investigations of human tRNA(3)(Lys). We use a straightforward set of NMR experiments to show that tRNA(3)(Lys) adopts a standard transfer ribonucleic acid tertiary structure in solution, and that Mg(2+) is required for this folding. The results underscore the power of NMR to reveal rapidly the conformation of RNAs.
View details for DOI 10.1016/j.febslet.2007.10.026
View details for Web of Science ID 000253487700022
View details for PubMedID 17963705
Rapid purification of RNAs using fast performance liquid chromatography (FPLC)
RNA-A PUBLICATION OF THE RNA SOCIETY
2007; 13 (2): 289-294
We present here an improved RNA purification method using fast performance liquid chromatography (FPLC) size-exclusion chromatography in place of denaturing polyacrylamide gel electrophoresis (PAGE). The method allows preparation of milligram quantities of pure RNA in a single day. As RNA oligonucleotides behave differently from globular proteins in the size-exclusion column, we present standard curves for RNA oligonucleotides of different lengths on both the Superdex 75 column and the Superdex 200 size-exclusion column. Using this approach, we can separate monomer from multimeric RNA species, purify the desired RNA product from hammerhead ribozyme reactions, and isolate refolded RNA that has aggregated after long-term storage. This methodology allows simple and rapid purification of RNA oligonucleotides for structural and biophysical studies.
View details for DOI 10.1261/rna.342607
View details for Web of Science ID 000243753500011
View details for PubMedID 17179067
Purification and characterization of transcribed RNAs using gel filtration chromatography
2007; 2 (12): 3270-3277
RNA synthesis using in vitro transcription by phage T7 RNA polymerase allows preparation of milligram quantities of RNA for biochemical, biophysical and structural investigations. Previous purification approaches relied on gel electrophoretic or gravity-flow chromatography methods. We present here a protocol for the in vitro transcription of RNAs and subsequent purification using fast-performance liquid chromatography. This protocol greatly facilitates production of RNA in a single day from transcription to purification.
View details for DOI 10.1038/nprot.2007.480
View details for Web of Science ID 000253140200026
View details for PubMedID 18079727
NMR investigation of HIV reverse transcription initiation
4th NATO Advanced-Study-Institute on Dynamics, Structure and Function of Biological Macromolecules
I O S PRESS. 2001: 187–192
View details for Web of Science ID 000170487100014
HIV-1 A-rich RNA loop mimics the tRNA anticodon structure
NATURE STRUCTURAL BIOLOGY
1998; 5 (12): 1033-1036
Interaction of HIV-1 genomic RNA and human tRNA(Lys)3 initiates viral reverse transcription. An adenosine-rich (A-rich) loop in HIV RNA mediates complex formation between tRNA and viral RNA. We have determined the structure of an A-rich loop oligonucleotide using nuclear magnetic resonance spectroscopy. The loop structure is stabilized by a noncanonical G-A pair and a U-turn motif, which leads to stacking of the conserved adenosines. The structure has similarity to the tRNA anticodon structure, and suggests possible mechanisms for its role in initiation of reverse transcription.
View details for Web of Science ID 000077284900005
View details for PubMedID 9846871
Structure of a conserved RNA component of the peptidyl transferase centre
NATURE STRUCTURAL BIOLOGY
1997; 4 (10): 775-778
The structure of a conserved hairpin loop involved in peptidyl-tRNA recognition by 50S ribosomal subunits has been solved by NMR. The loop is closed by a novel G-C base pair and presents guanine residues for RNA recognition.
View details for Web of Science ID A1997YA20300005
View details for PubMedID 9334738
Structure and function of ribosomal RNA
International Conference on the Structure and Function of the Ribosome
CANADIAN SCIENCE PUBLISHING, NRC RESEARCH PRESS. 1995: 997–1009
A refined model has been developed for the folding of 16S rRNA in the 30S subunit, based on additional constraints obtained from new experimental approaches. One set of constraints comes from hydroxyl radical footprinting of each of the individual 30S ribosomal proteins, using free Fe(2+)-EDTA complex. A second approach uses localized hydroxyl radical cleavage from a single Fe2+ tethered to unique positions on the surface of single proteins in the 30S subunit. This has been carried out for one position on the surface of protein S4, two on S17, and three on S5. Nucleotides in 16S rRNA that are essential for P-site tRNA binding were identified by a modification interference strategy. Ribosomal subunits were partially inactivated by chemical modification at a low level. Active, partially modified subunits were separated from inactive ones by binding 3'-biotinderivatized tRNA to the 30S subunits and captured with streptavidin beads. Essential bases are those that are unmodified in the active population but modified in the total population. The four essential bases, G926, 2mG966, G1338, and G1401 are a subset of those that are protected from modification by P-site tRNA. They are all located in the cleft of our 30S subunit model. The rRNA neighborhood of the acceptor end of tRNA was probed by hydroxyl radical probing from Fe2+ tethered to the 5' end of tRNA via an EDTA linker. Cleavage was detected in domains IV, V, and VI of 23S rRNA, but not in 5S or 16S rRNA. The sites were all found to be near bases that were protected from modification by the CCA end of tRNA in earlier experiments, except for a set of E-site cleavages in domain IV and a set of A-site cleavages in the alpha-sarcin loop of domain VI. In vitro genetics was used to demonstrate a base-pairing interaction between tRNA and 23S rRNA. Mutations were introduced at positions C74 and C75 of tRNA and positions 2252 and 2253 of 23S rRNA. Interaction of the CCA end of tRNA with mutant ribosomes was tested using chemical probing in conjunction with allele-specific primer extension. The interaction occurred only when there was a Watson-Crick pairing relationship between positions 74 of tRNA and 2252 of 23S rRNA. Using a novel chimeric in vitro reconstitution method, it was shown that the peptidyl transferase reaction depends on this same Watson-Crick base pair.
View details for Web of Science ID A1995UE60300027
View details for PubMedID 8722015
NMR ANALYSIS OF TRANSFER-RNA ACCEPTOR STEM MICROHELICES - DISCRIMINATOR BASE CHANGE AFFECTS TRANSFER-RNA CONFORMATION AT THE 3' END
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1994; 91 (24): 11467-11471
An important step in initiation of protein synthesis in Escherichia coli is the specific formylation of the initiator methionyl-tRNA (Met-tRNA) by Met-tRNA transformylase. The determinants for formylation are clustered mostly in the acceptor stem of the initiator tRNA. Here we use NMR spectroscopy to characterize the conformation of two RNA microhelices, which correspond to the acceptor stem of mutants of E. coli initiator tRNA and which differ only at the position corresponding to the "discriminator base" in tRNAs. One of the mutant tRNAs is an extremely poor substrate for Met-tRNA transformylase, whereas the other one is a much better substrate. We show that one microhelix forms a structure in which its 3'-ACCA sequence extends the stacking of the acceptor stem. The other microhelix forms a structure in which its 3'-UCCA sequence folds back such that the 3'-terminal A22 is in close proximity to G1. These results highlight the importance of the discriminator base in determining tRNA conformation at the 3' end. They also suggest a correlation between tRNA structure at the 3' end and its recognition by Met-tRNA transformylase.
View details for Web of Science ID A1994PU28500038
View details for PubMedID 7972085
A MONOCLONAL-ANTIBODY TO THE NH2-TERMINAL SEGMENT OF HUMAN IFN-GAMMA SELECTIVELY INTERFERES WITH THE ANTIPROLIFERATIVE ACTIVITY OF THE LYMPHOKINE
JOURNAL OF IMMUNOLOGY
1993; 150 (3): 1029-1035
To gain more information about the relationship between the structure of IFN-gamma and its activity, a peptide corresponding to a hydrophilic peak between amino acids 4 and 16 was used to immunize mice and generate mAb. mAb IGMB-15 reacts to both native and rIFN-gamma and neutralizes the antiproliferative activity of IFN-gamma without affecting its antiviral activity or its ability to up-regulate HLA-DR Ag expression. Moreover, we observed that mAb IGMB-15 was unable to inhibit the binding of radiolabeled IFN-gamma to its cellular receptor. These findings show that the NH2-terminal region may somehow be involved in the biologic activity of IFN-gamma. Besides, the capability of mAb IGMB-15 to inhibit the antiproliferative but not the antiviral activity of IFN-gamma in the same cell (HEp-2) suggests the presence of different elements involved in signal transduction, which may account for the multiple activities of the lymphokine.
View details for Web of Science ID A1993KJ09500034
View details for PubMedID 8423329
- Molecular Recognition in Water: New Receptors for Academic Derivatives J. Am. Chem. Soc. 1993: 797-798
NATURAL HUMAN-ANTIBODIES TO GAMMA INTERFERON INTERFERE WITH THE IMMUNOMODULATING ACTIVITY OF THE LYMPHOKINE
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1992; 89 (10): 4447-4451
Natural antibodies to gamma interferon (IFN-gamma) were found in patients suffering from different viral diseases and, at a lower titer, in healthy individuals. Such antibodies were affinity-purified and studied for their capability to interfere in vitro with the antiviral and immunomodulating activity of IFN-gamma. Data obtained show that these human anti-IFN-gamma antibodies have no inhibitory effect on the antiviral activity of IFN-gamma. On the contrary, they are able to inhibit the expression of Fc receptor sites and HLA-DR antigens induced by IFN-gamma on the U-937 cells, a human monocytoid/macrophage-derived cell line. These antibodies can also interfere in a mixed lymphocyte culture (MLC) with the proliferation of lymphocytes and the generation of cytotoxic lymphocytes. However, they showed only a moderate inhibitory effect on the cytotoxicity generated in MLC to K-562 cells. Human antibodies capable of interfering with the immunomodulating activities of IFN-gamma might open up a new field in clinical therapy for those diseases that carry evidence of activated cell-mediated immunity.
View details for Web of Science ID A1992HU97700047
View details for PubMedID 1316609
INHIBITION OF THE BIOLOGICAL-ACTIVITY OF HUMAN INTERFERON-GAMMA BY ANTIPEPTIDE ANTIBODIES
JOURNAL OF INTERFERON RESEARCH
1992; 12 (1): 49-54
To study the domain(s) responsible for the different biological activities of human interferon-gamma (HuIFN-gamma), rabbits were immunized with peptides corresponding to the five most hydrophilic amino acid sequences of the lymphokine. The resulting antisera were able to recognize both the immunizing peptide and the native protein. Antibodies to the carboxy-terminal region (amino acids 125-137) of HuIFN-gamma were able to interfere with the immunomodulating, antiviral, and antiproliferative activities of the lymphokine. Inhibition of the antiproliferative and antiviral activity was also observed using antibodies raised against the amino-terminal region (amino acids 4-16) and amino acids 80-95, respectively, but to a lesser extent than that observed with antibodies to the carboxyl terminus. The capability of these antibody preparations to partially interfere with only one of the lymphokine's biological activities might be explained by a mechanism of steric hindrance. The use of polyclonal antibodies allowed us to limit the presence of epitopes responsible for recombinant (r)HuIFN-gamma biological activities to the carboxy-terminal region.
View details for Web of Science ID A1992HE76000008
View details for PubMedID 1573282
PURIFICATION OF NATURAL HUMAN IFN-GAMMA ANTIBODIES
1991; 30 (1): 53-58
Natural antibodies to interferon gamma (IFN-gamma) were found in patients suffering from various viral infections, but also at weak titers in healthy individuals. In the present study we describe a one-step chromatographic procedure for the purification of the anti-IFN-gamma antibodies from human Ig preparations, using a recombinant IFN-gamma-coupled Sepharose CL4B affinity column. The antibodies to IFN-gamma were eluted from the column using 3 different methods without loss of immunological activity. They were found to be Ig, mostly of the IgG1 subclass, and, in the biological assay, to be able to neutralize the de novo expression of Fc receptor sites induced by IFN-gamma on U937 cells.
View details for Web of Science ID A1991GJ90300009
View details for PubMedID 1959942
NATURAL ANTIBODIES TO IFN-GAMMA IN MAN AND THEIR INCREASE DURING VIRAL-INFECTION
JOURNAL OF IMMUNOLOGY
1990; 144 (2): 685-690
Natural antibodies to IFN-gamma were found in healthy individuals ranging from newborn babies to adults and, at higher levels, in patients suffering from different viral infections. During a viral infection, the titer of anti-IFN-gamma antibodies was observed to be correlated with the stage of the disease. Antibodies specific to IFN-gamma were affinity purified both from sera taken from healthy individuals and sera from viral-infected patients, by using a rIFN-gamma-coupled CNBr-activated Sepharose 4B column. The antibodies were found to be of the IgG class, and maintained their ability to bind rIFN-gamma. They were then tested for neutralizing activity and none of the IgG preparations we analyzed impaired the antiviral activity of rIFN-gamma. This finding suggests that the antigenic determinants recognized by these antibodies on the IFN-gamma molecule are located outside the site, on the IFN-gamma molecule, responsible for its antiviral activity.
View details for Web of Science ID A1990CH29500041
View details for PubMedID 1688583