Role of an Active Site Adenine in Hairpin Ribozyme Catalysis

Kuzmin, Yaroslav I.; Costa, Carla P. Da; Cottrell, Joseph W.; Fedor, Martha J.

doi:10.1016/j.jmb.2005.04.005

Cited by 100 publications

(239 citation statements)

References 80 publications

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“…Crystal structures of the hairpin ribozyme (3) reveal the presence of guanine (G8) and adenine (A38) bases juxtaposed with the 2′-O and 5′-O, respectively, of the scissile phosphate, where they seem poised to act in general acid-base catalysis. This is consistent with the pH dependence of the reaction (4) and its variation with functional group modifications (5)(6)(7)(8).…”

supporting

confidence: 87%

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Wilson

Lü

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

120

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Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pK a ¼ 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5′ oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolatemodified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2′-O nucleophile by G638 and protonation of the 5′-O leaving group by A756. 5′-phosphorothiolate | RNA catalysis | nucleolytic ribozymes | catalytic mechanism R ibozyme-mediated catalysis is important for both RNA splicing and translation (1), yet its chemical origins are incompletely understood. The nucleolytic ribozymes bring about the site-specific cleavage or ligation of RNA, with an acceleration of a millionfold or greater. The intensively studied protein RNase A catalyses an identical cleavage reaction, and much evidence supports the hypothesis that each of these phosphoryl transfer reactions is subject to general acid-base catalysis. This mechanism requires a general base to deprotonate the attacking nucleophile, and a general acid to protonate the oxyanion leaving group (Fig. 1).The most common chemical entities implicated in RNA catalysis by the nucleolytic ribozymes are the nucleobases (2). Guanine appears to play a catalytic role in the hairpin, hammerhead, GlmS, and Varkud satellite (VS) ribozymes, adenine in the hairpin, and VS and cytosine in the hepatitis delta virus (HDV) ribozyme. Crystal structures of the hairpin ribozyme (3) reveal the presence of guanine (G8) and adenine (A38) bases juxtaposed with the 2′-O and 5′-O, respectively, of the scissile phosphate, where they seem poised to act in general acid-base catalysis. This is consistent with the pH dependence of the reaction (4) and its variation with functional group modifications (5-8).In its simplest active form, the VS ribozyme comprises five helices (II through VI) organized by two three-way junctions, which acts in trans upon a substrate stem loop (helix I) with an internal loop that contains the scissile phosphate (Fig. 1). The loop also contains the critical G638 (9). A756 (10-13) is contained within an internal loop in helix VI. While no crystal structure of the VS ribozyme has yet been solved, a small-angle X-ray scattering-derived model plac...

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supporting

confidence: 87%

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Wilson

Lü

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

120

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“…Water was not observed in the active site of previous 4WJ hairpin ribozyme structures, although solvent has been invoked to account for both structural and biochemical observations regarding the mechanism of action (10,(15)(16)(17). In earlier structural studies, it was noted that the N1 imino position of G8 appeared to function as an obligate proton donor to the 2'-oxygen of the nucleophile, and not as a proton acceptor as corroborated here for AP8 and DAP8.…”

Section: Implications For Water In the Active Sitementioning

confidence: 57%

“…The first entails acid/base catalysis involving G8 and/or A38 (14,23,52). The second calls for electrostatic stabilization involving localized nucleobase charge dissipation (15)(16)(17)23,53). Although X-ray crystallography cannot differentiate between these proposals, it can provide additional stereochemical information relevant to understanding the principles by which the enzyme promotes catalytically productive or non-productive states.…”

Section: Discussionmentioning

confidence: 99%

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,

et al. 2005

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The hairpin ribozyme requires functional group contributions from G8 to assist in phosphodiester bond cleavage. Previously, replacement of G8 by a series of nucleobase variants showed little effect on interdomain docking, but a 3-to 250-fold effect on catalysis. To identify G8 features that contribute to catalysis within the hairpin ribozyme active site, structures for five base variants were solved by X-ray crystallography in a resolution range between 2.3 to 2.7 Å. For comparison, a native all-RNA "G8" hairpin ribozyme structure was refined to 2.05 Å resolution. The native structure revealed a scissile bond angle (τ) of 158°, which is close to the requisite 180° 'in-line' geometry. Mutations G8(inosine), G8(diaminopurine), G8(aminopurine), G8(adenosine) and G8(uridine) folded properly, but exhibited non-ideal scissile bond geometries (τ ranging from 118° to 93°) that paralleled their diminished solution activities. A superposition ensemble of all structures, including a previously described hairpin ribozyme-vanadate complex, indicated the scissile bond can adopt a variety of conformations resulting from perturbation of the chemical environment, and provided a rationale for how the exocyclic amine of nucleobase 8 promotes productive, in-line geometry. Changes at position 8 also caused variations in the A−1 sugar pucker. In this regard, variants A8 and U8 appeared to represent non-productive ground-states in which their 2'-OH groups mimicked the pro-R, non-bridging oxygen of the vanadate transition-state complex. Finally, the results indicated that ordered water molecules bind near the 2'-hydroxyl of A−1, lending support to the hypothesis that solvent may play an important role in the reaction. † This work was supported by NIH Grant GM63162 to J.E.W. ‡ Protein Data Bank Codes for the reported structures: 1ZFR (G8), 1ZFT (G8I), 1ZFV (G8A), 1ZFX (G8U), 2BCY (G8AP), 2BB1 (G8DAP), 2BCZ (G8I/dA−1). § Present address: Rosalind Franklin School of Science and Medicine, Dept. Biochem. and Mol. Biol., 3333 Green Bay Road Rd., N. Chicago, IL 60064, USA * To whom correspondence should be addressed: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Box 712, Rochester, New York 14642 USA. Phone: 585 273-4516; Fax: 585 275-6007; Email: Joseph_Wedekind@URMC.Rochester.edu ∥ These authors contributed equally to this work.Supporting Information Available A stereo diagram of representative electron density maps is provided for the native and position 8 variants of this study (Figure 1). A diagram comparing the G8I/2'-OMe A−1 and G8I/2'-deoxy A−1 variants is also available (Figure 2). The method for HPLC composition analysis of AP8 and DAP8 crystals is reported, as well as elution profiles for the separated hairpin ribozyme strands (Figure 3). This information is provided free of charge at http://pubs.acs.org NIH Public Access The hairpin ribozyme is a small ribozyme whose family members catalyze a reversible, sitespecific phosphodiester bond cleavage reacti...

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“…In the latter case, one or more protons actually transfer to or from RNA functional group(s) in the rate-limiting step. Because we initiate the cleavage reaction by addition of MgCl 2 , it is also possible that Mg 2ϩ ions induce a conformational change in which a protonation or deprotonation event required for correct folding occurs; alternatively, as has been proposed for other ribozymes, the apparent titratable functional groups may be the general acid, general base, or electrostatic stabilizer in the chemical step of the reaction (6,19,(23)(24)(25)(26)(27).…”

Section: Resultsmentioning

confidence: 99%

“…For hairpin and hammerhead ribozymes, bell-shaped pH-rate curves are not experimentally observed. Apparent pK a values of Ϸ6 and Ϸ10 have been inferred for the hairpin ribozyme (23,26,51), and their involvement in general acid-base catalysis (26,51) or electrostatic stabilization (23) would provide estimates for k 1 of 10 4 to 10 5 min Ϫ1 . For the hammerhead ribozyme, two guanosine residues with pK a values of Ͼ9 have been proposed as candidates for the general acid and base (52, 53): assuming pK a values of 9.6 (the unshifted pK a of free guanosine) and extrapolating k obs from the log-linear portion of the pH-rate curve provides estimates for k 1 of 10 3 to 10 5 min Ϫ1 .…”

Section: Disrupting the Kissing Interaction Changes The Rate-limitingmentioning

confidence: 99%

Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme

Smith

Collins²

2007

Proc. Natl. Acad. Sci. U.S.A.

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A fast-cleaving version of the Varkud satellite ribozyme, called RG, shows an apparent cis-cleavage rate constant of 5 sec ؊1 , similar to the rates of protein enzymes that catalyze similar reactions. Here, we describe mutational, pH-rate, and kinetic solvent isotope experiments that investigate the identity and rate constant of the rate-limiting step in this reaction. Self-cleavage of RG exhibits a bell-shaped rate vs. pH profile with apparent pKas of 5.8 and 8.3, consistent with the protonation state of two nucleotides being important for the rate of cleavage. Cleavage experiments in heavy water (D2O) revealed a kinetic solvent isotope effect consistent with proton transfer in the rate-limiting step. A mutant RNA that disrupts a peripheral loop-loop interaction involved in RNA folding exhibits pH-and D2O-independent cleavage Ϸ10 3 -fold slower than wild type, suggesting that this mutant is limited by a different step than wild type. Substitution of adenosine 756 in the putative active-site loop with cytosine also decreases the cleavage rate Ϸ10 3 -fold, but the A756C mutant retains pH-and D2O-sensitivity similar to wild type, consistent with this mutant and wild type being limited by the chemical step of the reaction. These results suggest that the RG ribozyme provides a good experimental system to investigate the nature of fast, rate-limiting steps in a ribozyme cleavage reaction.kinetic solvent isotope effect ͉ kinetics ͉ Neurospora ͉ pH vs. rate S ince the discovery of RNA catalysis, several natural RNAs and many more RNA and DNA sequences obtained by in vitro selection have been shown to catalyze the cleavage and/or ligation of phosphodiester bonds, as well as other chemical activities. Recent work has begun to investigate the range of catalytic mechanisms used by ribozymes and to understand the similarities and differences with protein enzymes (1-3). The local environment of a folded RNA can shift the pK a of certain nucleobases by two or more pH units into the range where they could function as proton donors or acceptors in general acidbase catalysis, similar to histidines in their protein counterparts (4). Charged nucleobases could also participate in electrostatic stabilization in the transition state. Indeed, the bell-shaped rate vs. pH curves typical of protein enzymes that use general acid-base catalysis have been observed for certain hepatitis delta virus (HDV) ribozymes (5, 6) and Varkud satellite (VS) ribozyme (this study).Most previously characterized versions of the Neurospora VS ribozyme showed rather slow cis-or trans-cleavage apparent rate constants (k obs ) in the range of 1 min Ϫ1 or less and were only slightly affected by pH between pH 5.5 and 9.0 (7); however, a trans-ligating construct did exhibit pH dependence below pH 7.0 (8). Other observations have also raised the possibility that a protonated group could be involved in the rate-limiting step of a VS ribozyme reaction pathway. For example, Strobel and colleagues (9) observed pH-dependent rescue of a ligation reaction by using nucleo...

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Role of an Active Site Adenine in Hairpin Ribozyme Catalysis

Cited by 100 publications

References 80 publications

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,

Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme

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Role of an Active Site Adenine in Hairpin Ribozyme Catalysis

Cited by 100 publications

References 80 publications

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer,

Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme

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Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,