Topological crossing in the misfolded Tetrahymena ribozyme resolved by cryo-EM.
Proceedings of the National Academy of Sciences of the United States of America
2022; 119 (37): e2209146119
The Tetrahymena group I intron has been a key system in the understanding of RNA folding and misfolding. The molecule folds into a long-lived misfolded intermediate (M) invitro, which has been known to form extensive native-like secondary and tertiary structures but is separated by an unknown kinetic barrier from the native state (N). Here, we used cryogenic electron microscopy (cryo-EM) to resolve misfolded structures of the Tetrahymena L-21 ScaI ribozyme. Maps of three M substates (M1, M2, M3) and one N state were achieved from a single specimen with overall resolutions of 3.5 A, 3.8 A, 4.0 A, and 3.0 A, respectively. Comparisons of the structures reveal that all the M substates are highly similar to N, except for rotation of a core helix P7 that harbors the ribozyme's guanosine binding site and the crossing of the strands J7/3 and J8/7 that connect P7 to the other elements in the ribozyme core. This topological difference between the M substates and N state explains the failure of 5'-splice site substrate docking in M, supports a topological isomer model for the slow refolding of M to N due to a trapped strand crossing, and suggests pathways for M-to-N refolding.
View details for DOI 10.1073/pnas.2209146119
View details for PubMedID 36067294
Conserved Trigger Loop Histidine of RNA Polymerase II Functions as a Positional Catalyst Primarily through Steric Effects.
In all domains of life, multisubunit RNA polymerases (RNAPs) catalyze both the extension of mRNA transcripts by nucleotide addition and the hydrolysis of RNA, which enables proofreading by removal of misincorporated nucleotides. A highly conserved catalytic module within RNAPs called the trigger loop (TL) functions as the key controller of these activities. The TL is proposed to act as a positional catalyst of phosphoryl transfer and transcript cleavage via electrostatic and steric contacts with substrates in its folded helical form. The function of a near-universally conserved TL histidine that contacts NTP phosphates is of particular interest. Despite its exceptional conservation, substitutions of the TL His with Gln support efficient catalysis in bacterial and yeast RNAPs. Unlike bacterial TLs, which contain a nearby Arg, the TL His is the only acid-base catalyst candidate in the eukaryotic RNAPII TL. Nonetheless, replacement of the TL His with Leu is reported to support cell growth in yeast, suggesting that even hydrogen bonding and polarity at this position may be dispensable for efficient catalysis by RNAPII. To test how a TL His-to-Leu substitution affects the enzymatic functions of RNAPII, we compared its rates of nucleotide addition, pyrophosphorolysis, and RNA hydrolysis to those of the wild-type RNAPII enzyme. The His-to-Leu substitution slightly reduced rates of phosphoryl transfer with little if any effect on intrinsic transcript cleavage. These findings indicate that the highly conserved TL His is neither an obligate acid-base catalyst nor a polar contact for NTP phosphates but instead functions as a positional catalyst mainly through steric effects.
View details for DOI 10.1021/acs.biochem.1c00528
View details for PubMedID 34705427
Cryo-EM structures of full-length Tetrahymena ribozyme at 3.1 Å resolution.
Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution1-3. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure-function relationships4, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure-function relationships in ribozymes.
View details for DOI 10.1038/s41586-021-03803-w
View details for PubMedID 34381213
Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition.
2019; 74 (5): 951-965.e13
RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1, the scaffold of the nuclear compartment "paraspeckles," are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1. TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2, which partially protects its 3' UTR from miR-21-mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation.
View details for DOI 10.1016/j.molcel.2019.03.041
View details for PubMedID 31047794
View details for PubMedCentralID PMC6561722
Trigger loop of RNA polymerase is a positional, not acid-base, catalyst for both transcription and proofreading.
Proceedings of the National Academy of Sciences of the United States of America
2017; 114 (26): E5103-E5112
The active site of multisubunit RNA polymerases (RNAPs) is highly conserved from humans to bacteria. This single site catalyzes both nucleotide addition required for RNA transcript synthesis and excision of incorrect nucleotides after misincorporation as a proofreading mechanism. Phosphoryl transfer and proofreading hydrolysis are controlled in part by a dynamic RNAP component called the trigger loop (TL), which cycles between an unfolded loop and an α-helical hairpin [trigger helices (TH)] required for rapid nucleotide addition. The precise roles of the TL/TH in RNA synthesis and hydrolysis remain unclear. An invariant histidine residue has been proposed to function in the TH form as a general acid in RNA synthesis and as a general base in RNA hydrolysis. The effects of conservative, nonionizable substitutions of the TL histidine (or a neighboring TL arginine conserved in bacteria) have not yet been rigorously tested. Here, we report that glutamine substitutions of these residues, which preserve polar interactions but are incapable of acid-base chemistry, had little effect on either phosphoryl transfer or proofreading hydrolysis by Escherichia coli RNAP. The TL substitutions did, however, affect the backtracking of RNAP necessary for proofreading and potentially the reactivity of the backtracked nucleotide. We describe a unifying model for the function of the RNAP TL, which reconciles available data and our results for representative RNAPs. This model explains diverse effects of the TL basic residues on catalysis through their effects on positioning reactants for phosphoryl transfer and easing barriers to transcript backtracking, rather than as acid-base catalysts.
View details for DOI 10.1073/pnas.1702383114
View details for PubMedID 28607053
View details for PubMedCentralID PMC5495254