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Group I introns are catalytic RNAs that coordinate two consecutive transesterification reactions for self-splicing. To understand how the group I intron promotes catalysis and coordinates self-splicing reactions, we determine the structures of L-16 Tetrahymena ribozyme in complex with a 5′-splice site analog product and a 3′-splice site analog substrate using cryo-EM. We solve six conformations from a single specimen, corresponding to different splicing intermediates after the first ester-transfer reaction. The structures reveal dynamics during self-splicing, including large conformational changes of the internal guide sequence and the J5/4 junction as well as subtle rearrangements of active-site metals and the hydrogen bond formed between the 2′-OH group of A261 and the N2 group of guanosine substrate. These results help complete a detailed structural and mechanistic view of this paradigmatic group I intron undergoing the second step of self-splicing. The Tetrahymena ribozyme changes conformations to perform its self-splicing function. Here, the authors capture six structural snapshots of ribozyme during the second step of self-splicing from a single specimen, revealing the structural basis of how it promotes and coordinates self-splicing reactions.

Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution 1 – 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 relationships 4 , 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. Cryo-electron microscopy has been used to determine the structure of the Tetrahymena ribozyme (a catalytic RNA) at sufficiently high resolution to model side chains and metal ions.

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) in vitro, 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 Å, 3.8 Å, 4.0 Å, and 3.0 Å, 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.

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.

The stability and function of biomolecules are directly influenced by their myriad interactions with water. Nucleic acids are highly solvated and hence uniquely suited for the investigation of water in near-native conditions using cryogenic electron microscopy (cryo-EM). We determined two cryo-EM maps of Tetrahymena ribozyme 2.2 and 2.3 Å resolutions and automatically modeled and cross-validated water molecules and Mg2+ ions in the ribozyme core, revealing the extensive involvement of water in mediating RNA non-canonical interactions. Unexpectedly, in regions where we do not model ordered water, we observed highly similar densities in both cryo-EM maps. In many of these regions, the cryo-EM densities superimpose with complex water networks predicted by molecular dynamics (MD), supporting their assignment as water and suggesting a biophysical explanation for their elusiveness to conventional atomic coordinate modeling. The cryo-EM density can be further compared against various MD simulations enabling evaluation and comparison of the accuracy of various simulation methodologies.

The stability and function of biomolecules are directly influenced by their myriad interactions with water. In this study, we investigated water through cryogenic electron microscopy (cryo-EM) on a highly solvated molecule, the ribozyme, determined at 2.2 and 2.3 Å resolutions. By employing segmentation-guided water and ion modeling (SWIM), an approach combining resolvability and chemical parameters, we automatically modeled and cross-validated water molecules and Mg ions in the ribozyme core, revealing the extensive involvement of water in mediating RNA non-canonical interactions. Unexpectedly, in regions where SWIM does not model ordered water, we observed highly similar densities in both cryo-EM maps. In many of these regions, the cryo-EM densities superimpose with complex water networks predicted by molecular dynamics (MD), supporting their assignment as water and suggesting a biophysical explanation for their elusiveness to conventional atomic coordinate modeling. Our study demonstrates an approach to unveil both rigid and flexible waters that surround biomolecules through cryo-EM map densities, statistical and chemical metrics, and MD simulations.

Biomolecules rely on water and ions for stable folding, but these interactions are often transient, dynamic, or disordered and thus hidden from experiments and evaluation challenges that represent biomolecules as single, ordered structures. Here, we compare blindly predicted ensembles of water and ion structure to the cryo-EM densities observed around the ribozyme at 2.2-2.3 Å resolution, collected through target R1260 in the CASP16 competition. 26 groups participated in this solvation 'cryo-ensemble' prediction challenge, submitting over 350 million atoms in total, offering the first opportunity to compare blind predictions of dynamic solvent shell ensembles to cryo-EM density. Predicted atomic ensembles were converted to density through local alignment and these densities were compared to the cryo-EM densities using Pearson correlation, Spearman correlation, mutual information, and precision-recall curves. These predictions show that an ensemble representation is able to capture information of transient or dynamic water and ions better than traditional atomic models, but there remains a large accuracy gap to the performance ceiling set by experimental uncertainty. Overall, molecular dynamics approaches best matched the cryo-EM density, with blind predictions from bussilab_plain_md, SoutheRNA, bussilab_replex, coogs2, and coogs3 outperforming the baseline molecular dynamics prediction. This study indicates that simulations of water and ions can be quantitatively evaluated with cryo-EM maps. We propose that further community-wide blind challenges can drive and evaluate progress in modeling water, ions and other previously hidden components of biomolecular systems.

The stability and function of biomolecules are directly influenced by their myriad interactions with water. In this study, we investigated water through cryogenic electron microscopy (cryo-EM) on a highly solvated molecule, the Tetrahymena ribozyme, determined at 2.2 and 2.3 Angstrom resolutions. By employing segmentation-guided water and ion modeling (SWIM), an approach combining resolvability and chemical parameters, we automatically modeled and cross-validated water molecules and Mg2+ ions in the ribozyme core, revealing the extensive involvement of water in mediating RNA non-canonical interactions. Unexpectedly, in regions where SWIM does not model ordered water, we observed highly similar densities in both cryo-EM maps. In many of these regions, the cryo-EM densities superimpose with complex water networks predicted by molecular dynamics (MD), supporting their assignment as water and suggesting a biophysical explanation for their elusiveness to conventional atomic coordinate modeling. Our study demonstrates an approach to unveil both rigid and flexible waters that surround biomolecules through cryo-EM map densities, statistical and chemical metrics, and MD simulations.Competing Interest StatementThe authors have declared no competing interest.

Repeat-associated non-AUG (RAN) translation synthesizes protein in the absence of a cognate AUG start codon 1,2In frontotemporal dementia and amyotrophic lateral sclerosis, a GGGGCC (G4C2) repeat expansion in an intron of C9orf72 leads to synthesis of neurotoxic dipeptide-repeat proteins, underscoring the need to understand the mechanism of C9orf72 RAN translation1-5. RNA sequence and structure have been implicated, but how they direct C9orf72 RAN translation, particularly the rate-limiting, multi-step initiation phase, remains unclear6-10. We applied single-molecule biophysics to a reconstituted human translation initiation system and tracked fluorescently labeled ribosomes and initiation factors in real time. We show that RNA G4C2 repeats and sequence context alter initiation factor dynamics after ribosomal scanning, generating a kinetic bottleneck in the commitment to initiate at a near-cognate CUG start codon. Our model of C9orf72 RAN translation provides a mechanistic framework for how repeat expansions change underlying translation dynamics and may be broadly relevant to other disorders that involve RAN translation.