Identification of the catalytic subdomain of the VS ribozyme and evidence for remarkable sequence tolerance in the active site loop

We show here that the ribozyme domain of the Neurospora VS ribozyme consists of separable upper and lower subdomains. Deletion analysis demonstrates that the entire upper subdomain (helices III/IV/V) is dispensable for site-specific cleavage activity, providing experimental evidence that the active site is contained within the lower subdomain and within the substrate itself. We demonstrate an important role in cleavage activity for a region of helix VI called the 730 loop. Surprisingly, several loop sequences, sizes, and structures at this position can support site-specific cleavage, suggesting that a variety of non-Watson-Crick structures, rather than a specific loop structure, in this region of the ribozyme can contribute to formation of the active site.     

The role of phosphate groups in the VS ribozyme-substrate interaction

The VS ribozyme trans-cleavage substrate interacts with the catalytic RNA via tertiary interactions. To study the role of phosphate groups in the ribozyme–substrate interaction, 18 modified substrates were synthesized, where an epimeric phosphorothioate replaces one of the phosphate diester linkages. Sites in the stem–loop substrate where phosphorothioate substitution impaired reaction cluster in two regions. The first site is the scissile phosphate diester linkage and nucleotides downstream of this and the second site is within the loop region. The addition of manganese ions caused recovery of the rate of reaction for phosphorothioate substitutions between A621 and A622 and U631 and C632, suggesting that these two phosphate groups may serve as ligands for two metal ions. In contrast, significant manganese rescue was not observed for the scissile phosphate diester linkage implying that electrophilic catalysis by metal ions is unlikely to contribute to VS ribozyme catalysis. In addition, an increase in the reaction rate of the unmodified VS ribozyme was observed when a mixture of magnesium and manganese ions acted as the cofactor. One possible explanation for this effect is that the cleavage reaction of the VS ribozyme is rate limited by a metal dependent docking of the substrate on the ribozyme.     

Structure of the ribozyme substrate hairpin of Neurospora VS RNA: a close look at the cleavage site

The cleavage site of the Neurospora VS RNA ribozyme is located in a separate hairpin domain containing a hexanucleotide internal loop with an A-C mismatch and two adjacent G-A mismatches. The solution structure of the internal loop and helix la of the ribozyme substrate hairpin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The 2 nt in the internal loop, flanking the cleavage site, a guanine and adenine, are involved in two sheared G.A base pairs similar to the magnesium ion-binding site of the hammerhead ribozyme. Adjacent to the tandem G.A base pairs, the adenine and cytidine, which are important for cleavage, form a noncanonical wobble A+-C base pair. The dynamic properties of the internal loop and details of the high-resolution structure support the view that the hairpin structure represents a ground state, which has to undergo a conformational change prior to cleavage. Results of chemical modification and mutagenesis data of the Neurospora VS RNA ribozyme can be explained in context with the present three-dimensional structure.     

Ionization of a critical adenosine residue in the neurospora Varkud Satellite ribozyme active site

The Varkud Satellite (VS) ribozyme catalyzes a site-specific self-cleavage reaction that generates 5'-OH and 2',3'-cyclic phosphate products. Other ribozymes that perform an equivalent reaction appear to employ ionization of an active site residue, either to neutralize the negatively charged transition state or to act as a general acid-base catalyst. To test for important base ionization events in the VS ribozyme ligation reaction, we performed nucleotide analogue interference mapping (NAIM) with a series of ionization-sensitive adenosine and cytidine analogues. A756, a catalytically critical residue located within the VS active site, was the only nucleotide throughout the VS ribozyme that displayed the pH-dependent interference pattern characteristic of functional base ionization. We observed unique rescue of 8-azaadenosine (pK(a) 2.2) and purine riboside (pK(a) 2.1) interference at A756 at reduced reaction pH, suggestive of an ionization-specific effect. These results are consistent with protonation and/or deprotonation of A756 playing a direct role in the VS ribozyme reaction mechanism. In addition, NAIM experiments identified several functional groups within the RNA that play important roles in ribozyme folding and/or catalysis. These include residues in helix II, helix VI (730 loop), the II-III-VI and III-IV-V helix junctions, and loop V.     

NMR structure of the A730 loop of the Neurospora VS ribozyme: insights into the formation of the active site

The Neurospora VS ribozyme is a small nucleolytic ribozyme with unique primary, secondary and global tertiary structures, which displays mechanistic similarities to the hairpin ribozyme. Here, we determined the high-resolution NMR structure of a stem-loop VI fragment containing the A730 internal loop, which forms part of the active site. In the presence of magnesium ions, the A730 loop adopts a structure that is consistent with existing biochemical data and most likely reflects its conformation in the VS ribozyme prior to docking with the cleavage site internal loop. Interestingly, the A730 loop adopts an S-turn motif that is also present in loop B within the hairpin ribozyme active site. The S-turn appears necessary to expose the Watson-Crick edge of a catalytically important residue (A756) so that it can fulfill its role in catalysis. The A730 loop and the cleavage site loop of the VS ribozyme display structural similarities to internal loops found in the active site of the hairpin ribozyme. These similarities provided a rationale to build a model of the VS ribozyme active site based on the crystal structure of the hairpin ribozyme.     

4-thio-U cross-linking identifies the active site of the VS ribozyme

To identify nucleotides in or near the active site, we have used a circularly permuted version of the VS ribozyme capable of cleavage and ligation to incorporate a single photoactive nucleotide analog, 4-thio- uridine, immediately downstream of the scissile bond. Exposure to UV light produced two cross-linked RNAs, in which the 4-thio-uridine was cross-linked to A756 in the 730 loop of helix VI. The cross-links formed only under conditions that support catalytic activity, suggesting that they reflect functionally relevant conformations of the RNA. One of the cross-linked RNAs contains a lariat, indicative of intramolecular cross-linking in the ligated RNA; the other is a branched molecule in which the scissile phosphodiester bond is cleaved, but occupies the same site in the ribozyme-substrate complex. These are the two forms of the RNA expected to be the ground state structures on either side of the transition state. This localization of the active site is consistent with previous mutational, biochemical and biophysical data, and provides direct evidence that the cleavage site in helix I interacts with the 730 loop in helix VI.     

A pH controlled conformational switch in the cleavage site of the VS ribozyme substrate RNA

The VS ribozyme is a 154 nucleotide sequence found in certain natural strains of Neurospora. The RNA can be divided into a substrate and a catalytic domain. Here we present the solution structure of the substrate RNA that is cleaved in a trans reaction by the catalytic domain in the presence of Mg2+. The 30 nucleotide substrate RNA forms a compact helix capped by a flexible loop. The cleavage site bulge contains three non-canonical base-pairs, including an A+.C pair with a protonated adenine. This adenine (A622) is a pH controlled conformational switch that opens up the internal loop at higher pH. The possible significance of this switch for substrate recognition and cleavage is discussed.     

Formation of an active site in trans by interaction of two complete Varkud Satellite ribozymes

The complete VS ribozyme comprises seven helical segments, connected by three three-way RNA junctions. In the presence of Mg²⁺ ions, cleavage occurs within the internal loop of helix I. This requires the participation of a guanine (G638) within the helix I loop, and a remote adenine (A756) within an internal loop of helix VI. Previous structural studies have suggested that helix I docks into the fold of the remaining part of the ribozyme, bringing A756 and G638 close to the scissile phosphate to allow the cleavage reaction to proceed. We show here that while either A756C or G638A individually exhibit very low cleavage activity, a mixture of the two variants leads to cleavage of the A756C RNA, but not the G638A RNA. The rate of cleavage depends on the concentration of the VS G638A RNA, as expected for a bimolecular interaction. This regaining of cleavage activity by complementation indicates that helix I of one VS RNA can interact with another VS RNA molecule to generate a functional active site in trans.     

The A730 loop is an important component of the active site of the VS ribozyme

The core of the VS ribozyme comprises five helices, that act either in cis or in trans on a stem-loop substrate to catalyse site-specific cleavage. The structure of the 2-3-6 helical junction indicates that a cleft is formed between helices II and VI that is likely to serve as a receptor for the substrate. Detailed analysis of sequence variants suggests that the base bulges of helices II and VI play an architectural role. By contrast, the identity of the nucleotides in the A730 loop is very important for ribozyme activity. The base of A756 is particularly vital, and substitution by any other nucleotide or ablation of the base leads to a major reduction in cleavage rate. However, variants of A756 bind substrate efficiently, and are not defective in global folding. These results suggest that the A730 loop is an important component of the active site of the ribozyme, and that A756 could play a key role in catalysis.     

Requirement for canonical base pairing in the short pseudoknot structure of genomic hepatitis delta virus ribozyme

The tertiary structure of the 3′-cleaved product of the genomic hepatitis delta virus (HDV) ribozyme was solved by X-ray crystallographic analysis. In this structure, three single-stranded regions (SSrA, -B and -C) interact intricately with one another via hydrogen bonds between nucleotide bases, phosphate oxygens and 2′-OHs to form a nested double pseudoknot structure. Among these interactions, two Watson–Crick (W–C) base pairs, 726G–710C and 727G–709C, that form between SSrA and SSrC (P1.1) seem to be especially important for compact folding. To characterize the importance of these base pairs, ribozymes were subjected to in vitro selection from a pool of RNA molecules randomly substituted at positions 709, 710, 726 and 727. The results establish the importance of the two W–C base pairs for activity, although some mutants are active with one G–C base pair. In addition, the kinetic parameters were analyzed in all 16 combinations with two canonical base pairs. Comparison of variant ribozymes with the wild-type ribozyme reveals that the difference in reaction rates for these variants (ΔΔG^‡) is not simply accounted for by the differences in the stability of P1.1 (ΔΔG⁰₃₇). The role played by Mg²⁺ ions in formation of the P1.1 structure is also discussed.     

A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme

Kissing loops are tertiary structure elements that often play key roles in functional RNAs. In the Neurospora VS ribozyme, a kissing-loop interaction between the stem–loop I (SLI) substrate and stem–loop V (SLV) of the catalytic domain is known to play an important role in substrate recognition. In addition, this I/V kissing-loop interaction is associated with a helix shift in SLI that activates the substrate for catalysis. To better understand the role of this kissing-loop interaction in substrate recognition and activation by the VS ribozyme, we performed a thermodynamic characterization by isothermal titration calorimetry using isolated SLI and SLV stem–loops. We demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, with an energetic cost of 1.8–3 kcal/mol for the helix shift in SLI. The affinity of the preshifted SLI for SLV is remarkably high, the interaction being more stable by 7–8 kcal/mol than predicted for a comparable duplex containing three Watson–Crick base pairs. The structural basis of this remarkable stability is discussed in light of previous NMR studies. Comparative thermodynamic studies reveal that kissing-loop complexes containing 6–7 Watson–Crick base pairs are as stable as predicted from comparable RNA duplexes; however, those with 2–3 Watson–Crick base pairs are more stable than predicted. Interestingly, the stability of SLI/ribozyme complexes is similar to that of SLI/SLV complexes. Thus, the I/V kissing loop interaction represents the predominant energetic contribution to substrate recognition by the trans-cleaving VS ribozyme.     

Rearrangement of substrate secondary structure facilitates binding to the Neurospora VS ribozyme

The Neurospora VS ribozyme differs from other small, naturally occurring ribozymes in that it recognizes for trans cleavage or ligation a substrate that consists largely of a stem-loop structure. We have previously found that cleavage or ligation by the VS ribozyme requires substantial rearrangement of the secondary structure of stem-loop I, which contains the cleavage/ligation site. This rearrangement includes breaking the top base-pair of stem-loop I, allowing formation of a kissing interaction with loop V, and changing the partners of at least three other base-pairs within stem-loop I to adopt a conformation termed shifted. In the work presented, we have designed a binding assay and used mutational analysis to investigate the contribution of each of these structural changes to binding and ligation. We find that the loop I-V kissing interaction is necessary but not sufficient for binding and ligation. Constitutive opening of the top base-pair of stem-loop I has little, if any, effect on either activity. In contrast, the ability to adopt the shifted conformation of stem-loop I is a major determinant of binding: mutants that cannot adopt this conformation bind much more weakly than wild-type and mutants with a constitutively shifted stem-loop I bind much more strongly. These results implicate the adoption of the shifted structure of stem-loop I as an important process at the binding step in the VS ribozyme reaction pathway. Further investigation of features near the cleavage/ligation site revealed that sulphur substitution of the non-bridging phosphate oxygen atoms immediately downstream of the cleavage/ligation site, implicated in a putative metal ion binding site, significantly altered the cleavage/ligation equilibrium but did not perturb substrate binding significantly. This indicates that the substituted oxygen atoms, or an associated metal ion, affect a step that occurs after binding and that they influence the rates of cleavage and ligation differently.     

Structure, folding and activity of the VS ribozyme: importance of the 2-3-6 helical junction

The VS nucleolytic ribozyme has a core comprising five helices organized by two three-way junctions. The ribozyme can act in trans on a hairpin-loop substrate, with which it interacts via tertiary contacts. We have determined that one of the junctions (2-3-6) undergoes two-stage ion-dependent folding into a stable conformation, and have determined the global structure of the folded junction using long-range distance restraints derived from fluorescence resonance energy transfer. A number of sequence variants in the junction are severely impaired in ribozyme cleavage, and there is good correlation between changes in activity and alteration in the folding of junction 2-3-6. These studies point to a special importance of G and A nucleotides immediately adjacent to helix II, and comparison with a similar junction of known structure indicates that this could adopt a guanine-wedge structure. We propose that the 2-3-6 junction organizes important aspects of the structure of the ribozyme to facilitate productive association with the substrate, and suggest that this results in an interaction between the substrate and the A730 loop to create the active complex.     

The Varkud satellite ribozyme

The VS ribozyme is the largest nucleolytic ribozyme, for which there is no crystal structure to date. The ribozyme consists of five helical sections, organized by two three-way junctions. The global structure has been determined by solution methods, particularly FRET. The substrate stem-loop binds into a cleft formed between two helices, while making a loop-loop contact with another section of the ribozyme. The scissile phosphate makes a close contact with an internal loop (the A730 loop), the probable active site of the ribozyme. This loop contains a particularly critical nucleotide A756. Most changes to this nucleotide lead to three-orders of magnitude slower cleavage, and the Watson-Crick edge is especially important. NAIM experiments indicate that a protonated base is required at this position for the ligation reaction. A756 is thus a strong candidate for nucleobase participation in the catalytic chemistry.     

An important role of G638 in the cis-cleavage reaction of the Neurospora VS ribozyme revealed by a novel nucleotide analog incorporation method

We describe a chemical coupling procedure that allows joining of two RNAs, one of which contains a site-specific base analog substitution, in the absence of divalent ions. This method allows incorporation of nucleotide analogs at specific positions even into large, cis-cleaving ribozymes. Using this method we have studied the effects of substitution of G638 in the cleavage site loop of the VS ribozyme with a variety of purine analogs having different functional groups and pK(a) values. Cleavage rate versus pH profiles combined with kinetic solvent isotope experiments indicate an important role for G638 in proton transfer during the rate-limiting step of the cis-cleavage reaction.     

Rapid formation of a solvent-inaccessible core in the Neurospora Varkud satellite ribozyme

We have used hydroxyl radicals generated by decomposition of peroxynitrous acid to study Mg(2+)-dependent structure and folding of the Varkud satellite (VS) ribozyme. Protection from radical cleavage shows the existence of a solvent-inaccessible core, which includes nucleotides near two three-helix junctions, the kissing interaction between stem-loops I and V and other nucleotides, most of which have also been implicated as important for folding or activity. Kinetic folding experiments showed that the ribozyme folds very quickly, with the observed protections completely formed within 2 s of addition of MgCl(2). In mutants that disrupt the kissing interaction or entirely remove stem-loop I, which contains the cleavage site, nucleotides in the three-helix junctions and a subset of those elsewhere remain protected. Unlike smaller ribozymes, the VS ribozyme retains a significant amount of structure in the absence of its substrate. Protections that depend on proper interaction between the substrate and the rest ribozyme map to a region previously proposed as the active site of the ribozyme and along both sides of helix II, identifying candidate sites of docking for the substrate helix.     

Single VS ribozyme molecules reveal dynamic and hierarchical folding toward catalysis

Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two three-way junctions. Second, catalytic activity of the ribozyme is essential for replication of VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that the VS ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS ribozyme favor cleavage of the substrate downstream of the ribozyme core instead. This preference is expected to facilitate processing of the multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.     

Cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage

To monitor functionally important metal ions and possible cross talk in RNase P RNA mediated cleavage we studied cleavage of substrates, where the 2′OH at the RNase P cleavage site (at −1) and/or at position +73 had been replaced with a 2′ amino group (or 2′H). Our data showed that the presence of 2′ modifications at these positions affected cleavage site recognition, ground state binding of substrate and/or rate of cleavage. Cleavage of 2′ amino substituted substrates at different pH showed that substitution of Mg²⁺ by Mn²⁺ (or Ca²⁺), identity of residues at and near the cleavage site, and addition of C5 protein influenced the frequency of miscleavage at −1 (cleavage at the correct site is referred to as +1). From this we infer that these findings point at effects mediated by protonation/deprotonation of the 2′ amino group, i.e. an altered charge distribution, at the site of cleavage. Moreover, our data suggested that the structural architecture of the interaction between the 3′ end of the substrate and RNase P RNA influence the charge distribution at the cleavage site as well as the rate of cleavage under conditions where the chemistry is suggested to be rate limiting. Thus, these data provide evidence for cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage. We discuss the role metal ions might play in this cross talk and the likelihood that at least one functionally important metal ion is positioned in the vicinity of, and use the 2′OH at the cleavage site as an inner or outer sphere ligand.     

A guanine nucleobase important for catalysis by the VS ribozyme

A guanine (G638) within the substrate loop of the VS ribozyme plays a critical role in the cleavage reaction. Replacement by any other nucleotide results in severe impairment of cleavage, yet folding of the substrate is not perturbed, and the variant substrates bind the ribozyme with similar affinity, acting as competitive inhibitors. Functional group substitution shows that the imino proton on the N1 is critical, suggesting a possible role in general acid-base catalysis, and this in accord with the pH dependence of the reaction rate for the natural and modified substrates. We propose a chemical mechanism for the ribozyme that involves general acid-base catalysis by the combination of the nucleobases of guanine 638 and adenine 756. This is closely similar to the probable mechanism of the hairpin ribozyme, and the active site arrangements for the two ribozymes appear topologically equivalent. This has probably arisen by convergent evolution.     

Evidence that U2/U6 helix I promotes both catalytic steps of pre-mRNA splicing and rearranges in between these steps

During pre-mRNA splicing, the spliceosome must configure the substrate, catalyze 5′ splice site cleavage, reposition the substrate, and catalyze exon ligation. The highly conserved U2/U6 helix I, which adjoins sequences that define the reactive sites, has been proposed to configure the substrate for 5′ splice site cleavage and promote catalysis. However, a role for this helix at either catalytic step has not been tested rigorously and previous observations question its role at the catalytic steps. Through a comprehensive molecular genetic study of U2/U6 helix I, we found that weakening U2/U6 helix I, but not mutually exclusive structures, compromised splicing of a substrate limited at the catalytic step of 5′ splice site cleavage, providing the first compelling evidence that this helix indeed configures the substrate during 5′ splice site cleavage. Further, mutations that we proved weaken only U2/U6 helix I suppressed a mutation in PRP16, a DEAH-box ATPase required after 5′ splice site cleavage, providing persuasive evidence that helix I is destabilized by Prp16p and suggesting that this structure is unwound between the catalytic steps. Lastly, weakening U2/U6 helix I also compromised splicing of a substrate limited at the catalytic step of exon ligation, providing evidence that U2/U6 helix I reforms and functions during exon ligation. Thus, our data provide evidence for a fundamental and apparently dynamic role for U2/U6 helix I during the catalytic stages of splicing.