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Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme.

ABSTRACT: Twister is a recently discovered RNA motif that is estimated to have one of the fastest known catalytic rates of any naturally occurring small self-cleaving ribozyme. We determined the 4.1-Å resolution crystal structure of a twister sequence from an organism that has not been cultured in isolation, and it shows an ordered scissile phosphate and nucleotide 5' to the cleavage site. A second crystal structure of twister from Orzyza sativa determined at 3.1-Å resolution exhibits a disordered scissile phosphate and nucleotide 5' to the cleavage site. The core of twister is stabilized by base pairing, a large network of stacking interactions, and two pseudoknots. We observe three nucleotides that appear to mediate catalysis: a guanosine that we propose deprotonates the 2'-hydroxyl of the nucleotide 5' to the cleavage site and a conserved adenosine. We suggest the adenosine neutralizes the negative charge on a nonbridging phosphate oxygen atom at the cleavage site. The active site also positions the labile linkage for in-line nucleophilic attack, and thus twister appears to simultaneously use three strategies proposed for small self-cleaving ribozymes. The twister crystal structures (i) show its global structure, (ii) demonstrate the significance of the double pseudoknot fold, (iii) provide a possible hypothesis for enhanced catalysis, and (iv) illuminate the roles of all 10 highly conserved nucleotides of twister that participate in the formation of its small and stable catalytic pocket.

SUBMITTER: Eiler D

PROVIDER: S-EPMC4246988 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Pseudoknot Formation Seeds the Twister Ribozyme Cleavage Reaction Coordinate.

Project description:The twister RNA is a recently discovered nucleolytic ribozyme that is present in both bacteria and eukarya. While its biological role remains unclear, crystal structure analyses and biochemical approaches have revealed critical features of its catalytic mechanism. Here, we set out to explore dynamic aspects of twister RNA folding along the cleavage reaction coordinate. To do so, we have employed both bulk and single-molecule fluorescence resonance energy transfer (FRET) methods to investigate a set of twister RNAs with labels strategically positioned at communicating segments. The data reveal that folding of the central pseudoknot (T1), the most crucial structural determinant to promote cleavage, exhibits reversible opening and closing dynamics at physiological Mg2+ concentration. Uncoupled folding, in which T1 formation precedes structuring for closing of stem P1, was confirmed using pre-steady-state three-color smFRET experiments initiated by Mg2+ injection. This finding suggests that the folding path of twister RNA requires proper orientation of the substrate prior to T1 closure such that the U5-A6 cleavage site becomes embraced to achieve its cleavage competent conformation. We also find that the cleaved 3'-fragment retains its compacted pseudoknot fold, despite the absence of the phylogenetically conserved stem P1, rationalizing the poor turnover efficiency of the twister ribozyme.

| S-EPMC5697751 | biostudies-literature

Structure-based insights into self-cleavage by a four-way junctional twister-sister ribozyme.

Project description:Here we report on the crystal structure and cleavage assays of a four-way junctional twister-sister self-cleaving ribozyme. Notably, 11 conserved spatially separated loop nucleotides are brought into close proximity at the ribozyme core through long-range interactions mediated by hydrated Mg2+ cations. The C62-A63 step at the cleavage site adopts a splayed-apart orientation, with flexible C62 directed outwards, whereas A63 is directed inwards and anchored by stacking and hydrogen-bonding interactions. Structure-guided studies of key base, sugar, and phosphate mutations in the twister-sister ribozyme, suggest contributions to the cleavage chemistry from interactions between a guanine at the active site and the non-bridging oxygen of the scissile phosphate, a feature found previously also for the related twister ribozyme. Our four-way junctional pre-catalytic structure differs significantly in the alignment at the cleavage step (splayed-apart vs. base-stacked) and surrounding residues and hydrated Mg2+ ions relative to a reported three-way junctional pre-catalytic structure of the twister-sister ribozyme.

| S-EPMC5660989 | biostudies-literature

RNase footprinting demonstrates antigenomic hepatitis delta virus ribozyme structural rearrangement as a result of self-cleavage reaction.

Project description:BACKGROUND: Hepatitis delta virus (HDV) is a satellite virus of hepatitis B. During viral replication the 1700-nucleotide-long genomic RNA and its complement, the antigenomic RNA, undergo self-cleavage catalyzed by internal ribozyme motifs that are essential for propagation of the virus in vivo. These self-cleavage activities are provided by 85-nucleotide-long sequence elements, the genomic and antigenomic forms of HDV ribozyme. Recently four permuted variants of the antigenomic HDV cis-ribozyme with a self-cleavage site located at the 5' proximity, in the middle, or nearby the 3' end of the molecule were constructed and synthesized. These constructs exhibit equal activity, a bi-phasic kinetics of self-cleavage reaction and reaction products with low and high stability. We have used ribonuclease probing to footprint the structures of uncleaved and post-cleaved forms of the antigenomic HDV ribozymes in solution. Uncleaved ribozymes, associated and individual products of the self-cleavage reaction were analyzed using ribonuclease and Fe(II)-EDTA protection assays to reveal the differences in the structure of pre- and post-cleaved antigenomic HDV ribozyme in solution. FINDINGS: Our findings demonstrate that a significant conformational change accompanies catalysis in the antigenomic HDV ribozyme in solution, in contrast to minor conformational switch observed in crystals of the genomic form. This study indicates that changes in the structure of stem P1 and stem P4 are minor, those of the region ascribed to stem P2, stem P3 and loop l3 are dramatic, while stem P1.1 results from the self-cleavage reaction. CONCLUSION: Our data agree with the structure of post-cleaved and disagree with that of pre-cleaved forms of HDV ribozyme published elsewhere.

| S-EPMC2518280 | biostudies-literature

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation.

Project description:The majority of structural efforts addressing RNA's catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a lambda-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme- and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions.

| S-EPMC4692364 | biostudies-literature

In-line alignment and Mg²⁺ coordination at the cleavage site of the env22 twister ribozyme.

Project description:Small self-cleaving nucleolytic ribozymes contain catalytic domains that accelerate site-specific cleavage/ligation of phosphodiester backbones. We report on the 2.9-Å crystal structure of the env22 twister ribozyme, which adopts a compact tertiary fold stabilized by co-helical stacking, double-pseudoknot formation and long-range pairing interactions. The U-A cleavage site adopts a splayed-apart conformation with the modelled 2'-O of U positioned for in-line attack on the adjacent to-be-cleaved P-O5' bond. Both an invariant guanosine and a Mg(2+) are directly coordinated to the non-bridging phosphate oxygens at the U-A cleavage step, with the former positioned to contribute to catalysis and the latter to structural integrity. The impact of key mutations on cleavage activity identified an invariant guanosine that contributes to catalysis. Our structure of the in-line aligned env22 twister ribozyme is compared with two recently reported twister ribozymes structures, which adopt similar global folds, but differ in conformational features around the cleavage site.

| S-EPMC4373348 | biostudies-literature

Coupling of fast and slow modes in the reaction pathway of the minimal hammerhead ribozyme cleavage.

Project description:By employing classical molecular dynamics, correlation analysis of coupling between slow and fast dynamical modes, and free energy (umbrella) sampling using classical as well as mixed quantum mechanics molecular mechanics force fields, we uncover a possible pathway for phosphoryl transfer in the self-cleaving reaction of the minimal hammerhead ribozyme. The significance of this pathway is that it initiates from the minimal hammerhead crystal structure and describes the reaction landscape as a conformational rearrangement followed by a covalent transformation. The delineated mechanism is catalyzed by two metal (Mg(2+)) ions, proceeds via an in-line-attack by CYT 17 O2' on the scissile phosphorous (ADE 1.1 P), and is therefore consistent with the experimentally observed inversion configuration. According to the delineated mechanism, the coupling between slow modes involving the hammerhead backbone with fast modes in the cleavage site appears to be crucial for setting up the in-line nucleophilic attack.

| S-EPMC1965431 | biostudies-literature

Structural Basis for Substrate Helix Remodeling and Cleavage Loop Activation in the Varkud Satellite Ribozyme.

Project description:The Varkud satellite (VS) ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the ribozyme, resulting in a rearrangement of the substrate helix register into a so-called "shifted" conformation that is critical for substrate binding and activation. We report a 3.3 Å crystal structure of the complete ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and ribozyme cleavage.

| S-EPMC5929484 | biostudies-literature

A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.

Project description:Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a (13) C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg(2+) ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn(2+) or Cd(2+) accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6?Å X-ray structure of a 2'-OCH3 -U5 modified twister ribozyme.

| S-EPMC4715771 | biostudies-literature

QM/MM studies of hairpin ribozyme self-cleavage suggest the feasibility of multiple competing reaction mechanisms.

Project description:The hairpin ribozyme is a prominent member of small ribozymes since it does not require metal ions to achieve catalysis. Guanine 8 (G8) and adenine 38 (A38) have been identified as key participants in self-cleavage and -ligation. We have carried out hybrid quantum-mechanical/molecular mechanical (QM/MM) calculations to evaluate the energy along several putative reaction pathways. The error of our DFT description of the QM region was tested and shown to be ~1 kcal/mol. We find that self-cleavage of the hairpin ribozyme may follow several competing microscopic reaction mechanisms, all with calculated activation barriers in good agreement with those from experiment (20-21 kcal/mol). The initial nucleophilic attack of the A-1(2'-OH) group on the scissile phosphate is predicted to be rate-limiting in all these mechanisms. An unprotonated G8(-) (together with A38H(+)) yields a feasible activation barrier (20.4 kcal/mol). Proton transfer to a nonbridging phosphate oxygen also leads to feasible reaction pathways. Finally, our calculations consider thio-substitutions of one or both nonbridging oxygens of the scissile phosphate and predict that they have only a negligible effect on the reaction barrier, as observed experimentally.

| S-EPMC3223549 | biostudies-literature

Cellular Small Molecules Contribute to Twister Ribozyme Catalysis.

Project description:The number of self-cleaving small ribozymes has increased sharply in recent years. Advances have been made in describing these ribozymes in terms of four catalytic strategies: ? describes in-line attack, ? describes neutralization of the nonbridging oxygens, ? describes activation of the nucleophile, and ? describes stabilization of the leaving group. Current literature presents the rapid self-cleavage of the twister ribozyme in terms of all four of these classic catalytic strategies. Herein, we describe the nonspecific contribution of small molecules to ribozyme catalysis. At biological pH, the rate of the wild-type twister ribozyme is enhanced up to 5-fold in the presence of moderate buffer concentrations, similar to the 3-5-fold effects reported previously for buffer catalysis for protein enzymes. We observe this catalytic enhancement not only with standard laboratory buffers, but also with diverse biological small molecules, including imidazole, amino acids, and amino sugars. Brønsted plots suggest that small molecules assist in proton transfer, most likely with ? catalysis. Cellular small molecules provide a simple way to overcome the limited functional diversity of RNA and have the potential to participate in the catalytic mechanisms of many ribozymes in vivo.

| S-EPMC6472948 | biostudies-literature

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