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.     

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.     

Role of an active site adenine in hairpin ribozyme catalysis

The hairpin ribozyme is a small catalytic RNA that accelerates reversible cleavage of a phosphodiester bond. Structural and mechanistic studies suggest that divalent metals stabilize the functional structure but do not participate directly in catalysis. Instead, two active site nucleobases, G8 and A38, appear to participate in catalytic chemistry. The features of A38 that are important for active site structure and chemistry were investigated by comparing cleavage and ligation reactions of ribozyme variants with A38 modifications. An abasic substitution of A38 reduced cleavage and ligation activity by 14,000-fold and 370,000-fold, respectively, highlighting the critical role of this nucleobase in ribozyme function. Cleavage and ligation activity of unmodified ribozymes increased with increasing pH, evidence that deprotonation of some functional group with an apparent pK(a) value near 6 is important for activity. The pH-dependent transition in activity shifted by several pH units in the basic direction when A38 was substituted with an abasic residue, or with nucleobase analogs with very high or low pK(a) values that are expected to retain the same protonation state throughout the experimental pH range. Certain exogenous nucleobases that share the amidine group of adenine restored activity to abasic ribozyme variants that lack A38. The pH dependence of chemical rescue reactions also changed according to the intrinsic basicity of the rescuing nucleobase, providing further evidence that the protonation state of the N1 position of purine analogs is important for rescue activity. These results are consistent with models of the hairpin ribozyme catalytic mechanism in which interactions with A38 provide electrostatic stabilization to the transition state.     

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.     

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.     

Analysis of the cleavage reaction of a trans-acting human hepatitis delta virus ribozyme.

The cleavage reaction catalyzed by the trans -acting genomic ribozyme of human hepatitis delta virus (HDV) was analyzed with a 13mer substrate (R13) and thio-substituted [SR13(Rp) and SR13(Sp)] substrates under single-turnover conditions. The cleavage of RNA by the trans -acting HDV ribozyme proceeded as a first order reaction. The logarithm of the rate of cleavage (kclv) increased linearly (with a slope of approximately 1) between pH 4.0 and 6.0, an indication that a single deprotonation reaction occurred. This result suggests that kclv reflects the rate of the chemical cleavage step, at least around pH 5. The amount of active complex with the SR13(Sp) substrate was almost as large as with R13 (60-80%), whereas the amount of the corresponding active complex formed with the SR13(Rp) substrate was, at most, 20% of this value (with 0.5-100 mM Mg2+ions) at pH 5.0. Nonetheless, the value of kclv for all substrates was almost the same (0.4-0.5 min-1). Neither a 'thio effect' nor a 'Mn2+rescue effect' were observed. These results suggest that Mg2+ions do not interact with pro-R oxygen directly but are essential to the formation of the active complex of the ribozyme and its substrate.     

Reaction pathway of the trans-acting hepatitis delta virus ribozyme: a conformational change accompanies catalysis.

The hepatitis delta virus (HDV), an infectious human pathogen and satellite of hepatitis B virus, leads to intensified disease symptoms, including progression to liver cirrhosis. Both the circular RNA genome of HDV and its complementary antigenome contain the same cis-cleaving catalytic RNA motif that plays a crucial role in virus replication. Previously, the high-resolution crystal structure of the product form of a cis-acting genomic HDV ribozyme has been determined, while a trans-acting version of the ribozyme was used to dissect the cleavage reaction pathway. Using fluorescence resonance energy transfer (FRET) on a synthetic trans-cleaving form of the ribozyme, we are able to directly observe substrate binding (at a rate constant k(on) of 7.8 x 10(6) M(-1) min(-1) at pH 7.5, 11 mM MgCl(2), and 25 degrees C) and dissociation (at 0.34 min(-1)). Steady-state and time-resolved FRET experiments in solution and in nondenaturing gels reveal that the substrate (precursor) complex is slightly more compact (by approximately 3 A) than the free ribozyme, yet becomes significantly extended (by approximately 15 A) upon cleavage and product complex formation. We also find that trans cleavage is characterized by a high transition-state entropy (-26 eu). We propose that the significant global conformational change that we observe between the precursor and product structures occurs on the reaction trajectory into a constrained product complex-like transition state. Our observations may present the structural basis of the recently described utilization of intrinsic substrate binding energy to the overall catalytic rate enhancement by the trans-acting HDV ribozyme.     

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.     

Catalytically active geometry in the reversible circularization of 'mini-monomer' RNAs derived from the complementary strand of tobacco ringspot virus satellite RNA.

The less abundant polarity of the satellite RNA of tobacco ringspot virus, designated sTobRV(-)RNA, contains a ribozyme and its substrate. We demonstrate that the ribozyme can catalyze the ligation of substrate cleavage products and that oligoribonucleotides, termed 'mini-monomers' and containing little more than covalently attached ribozyme and substrate cleavage products, circularized spontaneously, efficiently and reversibly. The kinetics of ligation and cleavage of one such mini-monomer was consistent with a simple unimolecular reaction at some temperatures. Evidence suggests that the circular ligation product includes a 5 bp stem that is connected to a 4 bp stem by a bulge loop. Reduction of the bulge loop to one nt is expected to place the 4 and 5 bp helices in a nearly coaxial, rather than an angled or parallel, orientation. Such molecules did not circularize in a unimolecular reaction but did when incubated with second, trans-acting oligoribonucleotides that had either the original or a substituted 4 bp helix. These results suggest that a bulge loop that is too small prevents formation of geometry essential for unimolecular ligation. We suggest the term 'paperclip' to represent the arrangement of RNA strands in the region of sTobRV(-)RNA that participates in the cleavage and ligation reactions.     

Role of an active site guanine in hairpin ribozyme catalysis probed by exogenous nucleobase rescue

The hairpin ribozyme is a small catalytic RNA with reversible phosphodiester cleavage activity. Biochemical and structural studies exclude a requirement for divalent metal cation cofactors and implicate one active site nucleobase in particular, G8, in the catalytic mechanism. Our previous work demonstrated that the cleavage activity that is lost when G8 is replaced by an abasic residue is restored when certain nucleobases are provided in solution. The specificity and pH dependence of exogenous nucleobase rescue were consistent with several models of the rescue mechanism, including general acid base catalysis, electrostatic stabilization of negative charge in the transition state or a requirement for protonation to facilitate exogenous nucleobase binding. Detailed analyses of exogenous nucleobase rescue for both cleavage and ligation reactions now allow us to refine models of the rescue mechanism. Activity increased with increasing pH for both unmodified ribozyme reactions and unrescued reactions of abasic variants lacking G8. This similarity in pH dependence argues against a role for G8 as a general base catalyst, because G8 deprotonation could not be responsible for the pH-dependent transition in the abasic variant. Exogenous nucleobase rescue of both cleavage and ligation activity increased with decreasing pH, arguing against a role for rescuing nucleobases in general acid catalysis, because a nucleobase that contributes general acid catalysis in the cleavage pathway should provide general base catalysis in ligation. Analysis of the concentration dependence of cytosine rescue at high and low pH demonstrated that protonation promotes catalysis within the nucleobase-bound ribozyme complex but does not stabilize nucleobase binding in the ground state. These results support an electrostatic stabilization mechanism in which exogenous nucleobase binding counters negative charge that develops in the transition state.     

Kinetic characterization of the first step of the ribozyme-catalyzed trans excision-splicing reaction

Group I introns catalyze the self-splicing reaction, and their derived ribozymes are frequently used as model systems for the study of RNA folding and catalysis, as well as for the development of non-native catalytic reactions. Utilizing a group I intron-derived ribozyme from Pneumocystis carinii, we previously reported a non-native reaction termed trans excision-splicing (TES). In this reaction, an internal segment of RNA is excised from an RNA substrate, resulting in the covalent reattachment of the flanking regions. TES proceeds through two consecutive phosphotransesterification reactions, which are similar to the reaction steps of self-splicing. One key difference is that TES utilizes the 3'-terminal guanosine of the ribozyme as the first-step nucleophile, whereas self-splicing utilizes an exogenous guanosine. To further aid in our understanding of ribozyme reactions, a kinetic framework for the first reaction step (substrate cleavage) was established. The results demonstrate that the substrate binds to the ribozyme at a rate expected for simple helix formation. In addition, the rate constant for the first step of the TES reaction is more than one order of magnitude lower than the analogous step in self-splicing. Results also suggest that a conformational change, likely similar to that in self-splicing, exists between the two reaction steps of TES. Finally, multiple turnover is curtailed because dissociation of the cleavage product is slower than the rate of chemistry.     

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.     

Hairpin ribozyme-catalyzed ligation in water-alcohol solutions

The hairpin ribozyme (HPR) is a naturally existing RNA that catalyzes site-specific RNA cleavage and ligation. At 37 degrees C and in the presence of divalent metal ions (M(2+)), the HPR efficiently cleaves RNA substrates in trans. Here, we show that the HPR can catalyze efficient M(2+)-independent ligation in trans in aqueous solutions containing any of several alcohols, including methanol, ethanol, and isopropanol, and millimolar concentrations of monovalent cations. Ligation proceeds most efficiently in 60% isopropanol at 37 degrees C, whereas the reverse (cleavage) reaction is negligible under these conditions. We suggest that dehydration of the RNA is the key factor promoting HPR activity in water- alcohol solutions. Alcohol-induced ribozyme ligation may have practical applications.     

Design of hairpin ribozyme variants with improved activity for poorly processed substrates

Application of ribozymes for knockdown of RNA targets requires the identification of suitable target sites according to the consensus sequence. For the hairpin ribozyme, this was originally defined as Y?2 N?1 *G+1 U+2 Y+3 B+?, with Y = U or C, and B = U, C or G, and C being the preferred nucleobase at positions -2 and +4. In the context of development of ribozymes for destruction of an oncogenic mRNA, we have designed ribozyme variants that efficiently process RNA substrates at U?2 G?1 *G+1 U+2 A+3 A+? sites. Substrates with G?1 *G+1 U+2 A+3 sites were previously shown to be processed by the wild-type hairpin ribozyme. However, our study demonstrates that, in the specific sequence context of the substrate studied herein, compensatory base changes in the ribozyme improve activity for cleavage (eight-fold) and ligation (100-fold). In particular, we show that A+3 and A+? are well tolerated if compensatory mutations are made at positions 6 and 7 of the ribozyme strand. Adenine at position +4 is neutralized by G? →U, owing to restoration of a Watson-Crick base pair in helix 1. In this ribozyme-substrate complex, adenine at position +3 is also tolerated, with a slightly decreased cleavage rate. Additional substitution of A? with uracil doubled the cleavage rate and restored ligation, which was lost in variants with A?, C? and G?. The ability to cleave, in conjunction with the inability to ligate RNA, makes these ribozyme variants particularly suitable candidates for RNA destruction.     

Efficient ligation of the Schistosoma hammerhead ribozyme

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by an approximately 2000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2 to 3 at physiological Mg2+ ion concentrations (0.1-1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme.     

A chemo-genetic approach for the study of nucleobase participation in nucleolytic ribozymes

A novel chemo-genetic approach for the analysis of general acid-base catalysis by nucleobases in ribozymes is reviewed. This involves substitution of a C-nucleoside with imidazole in place of a natural nucleobase. The Varkud satellite ribozyme in which the nucleobase at the critical 756 position has been replaced by imidazole is active in both cleavage and ligation reactions. Similarly, a modified hairpin ribozyme with the nucleobase at position 8 substituted by imidazole is active in cleavage and ligation reactions. Although the rates are lower than those of the natural ribozymes, they are significantly greater than other variants at these positions. The dependence of the hairpin ribozyme reaction rates on pH has been studied. Both cleavage and ligation reactions display a bell-shaped pH dependence, consistent with general acid-base catalysis involving the nucleotide at position 8.     

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.     

Cross-ligation and exchange reactions catalyzed by hairpin ribozymes.

The negative strand of the satellite RNA of tobacco ringspot virus (sTobRV(-)) contains a hairpin catalytic domain that shows self-cleavage and self-ligation activities in the presence of magnesium ions. We describe here that the minimal catalytic domain can catalyze a cross-ligation reaction between two kinds of substrates in trans. The cross-ligated product increased when the reaction temperature was decreased during the reaction from 37 degrees C to 4 degrees C. A two-stranded hairpin ribozyme, divided into two fragments between G45 and U46 in a hairpin loop, showed higher ligation activity than the nondivided ribozyme. The two stranded ribozyme also catalyzed an exchange reaction of the 3'-portion of the cleavage site.     

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.     

Mutational inhibition of ligation in the hairpin ribozyme: substitutions of conserved nucleobases A9 and A10 destabilize tertiary structure and selectively promote cleavage

The hairpin ribozyme acts as a reversible, site-specific endoribonuclease that ligates much more rapidly than it cleaves cognate substrate. While the reaction pathway for ligation is the reversal of cleavage, little is known about the atomic and electrostatic details of the two processes. Here, we report the functional consequences of molecular substitutions of A9 and A10, two highly conserved nucleobases located adjacent to the hairpin ribozyme active site, using G, C, U, 2-aminopurine, 2,6-diaminopurine, purine, and inosine. Cleavage and ligation kinetics were analyzed, tertiary folding was monitored by hydroxyl radical footprinting, and interdomain docking was studied by native gel electrophoresis. We determined that nucleobase substitutions that exhibit significant levels of interference with tertiary folding and interdomain docking have relatively large inhibitory effects on ligation rates while showing little inhibition of cleavage. Indeed, one variant, A10G, showed a fivefold enhancement of cleavage rate and no detectable ligation, and we suggest that this property may be uniquely well suited to intracellular targeted RNA cleavage applications. Results support a model in which formation of a kinetically stable tertiary structure is essential for ligation of the hairpin ribozyme, but is not necessary for cleavage.