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Nucleobase-Mediated General Acid-Base Catalysis in the Varkud Satellite Ribozyme

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Nucleobase-Mediated General Acid-Base Catalysis in the Varkud Satellite Ribozyme Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme Timothy J. Wilsona, Nan-Sheng Lib, Jun Lub, John K. Frederiksenb,2, Joseph A. Piccirillib,1, and David M. J. Lilleya,1 aCancer Research United Kingdom Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom; and bThe University of Chicago Center for Integrative Science, Departments of Biochemistry and Molecular Biology and Chemistry, 929 East 57th Street, Room W406, Chicago, IL 60637 Edited by Olke C. Uhlenbeck, Northwestern University, Evanston, IL, and approved May 19, 2010 (received for review March 30, 2010) 2þ Existing evidence suggests that the Varkud satellite (VS) ribozyme At high concentrations of Mg ions, the pH dependence of accelerates the cleavage of a specific phosphodiester bond using the cleavage reaction of the trans-acting VS ribozyme is bell general acid-base catalysis. The key functionalities are the nucleo- shaped, fitting a model involving proton transfers in the transition K ¼ 5 2 bases of adenine 756 in helix VI of the ribozyme, and guanine 638 state with participating groups of p a . and 8.4 (9). Substitu- in the substrate stem loop. This results in a bell-shaped dependence tion of G638 by diaminopurine shifts the pH profile, correspond- of reaction rate on pH, corresponding to groups with pK ¼ 5.2 and K a ing to new p a values of 4.6 and 5.6. A plausible mechanism 8.4. However, it is not possible from those data to determine which involves general acid-base catalysis by A756 and G638, but it nucleobase is the acid, and which the base. We have therefore is not possible to determine from the pH dependence which ′ made substrates in which the 5 oxygen of the scissile phosphate nucleobase acts as the acid, and which the base. The alternatives is replaced by sulfur. This labilizes the leaving group, removing the predict identical pH profiles. Smith et al. have suggested a requirement for general acid catalysis. This substitution restores resolution of this ambiguity, based on the relationship between full activity to the highly impaired A756G ribozyme, consistent K ionic environment and nucleobase p a (15), but there is no with general acid catalysis by A756 in the unmodified ribozyme. direct evidence enabling the assignment of function to specific The pH dependence of the cleavage of the phosphorothiolate- nucleobases. modified substrates is consistent with general base catalysis by A similar ambiguity existed for the HDV ribozyme, where the nucleobase at position 638. We conclude that cleavage of the critical functionalities are a cytosine nucleobase (16–18) and a substrate by the VS ribozyme is catalyzed by deprotonation of metal ion-bound water (2). Assignment of the general acid and the 2′-O nucleophile by G638 and protonation of the 5′-O leaving group by A756. base was both difficult and controversial. The distinction was made by the introduction of a 5′-phosphorothiolate (5′-PS) substitution ′ 5′-phosphorothiolate ∣ RNA catalysis ∣ nucleolytic ribozymes ∣ at the scissile phosphate (19). The 5 sulfur atom is a much better catalytic mechanism leaving group than oxygen and therefore no longer requires protonation by a general acid. Thus alterations to the ribozyme ibozyme-mediated catalysis is important for both RNA that impair the function of the general acid (so inhibiting cleavage ′ Rsplicing and translation (1), yet its chemical origins are incom- of the oxy substrate) should have little effect on cleavage of a 5 -PS- pletely understood. The nucleolytic ribozymes bring about the containing substrate. Moreover, the pH dependence of the site-specific cleavage or ligation of RNA, with an acceleration cleavage rate of the 5′-PS substrate should reflect the deprotona- of a millionfold or greater. The intensively studied protein tion of the base alone. We have therefore synthesized VS ribozyme RNase A catalyses an identical cleavage reaction, and much substrates containing a 5′-PS linkage at the cleavage site. The evidence supports the hypothesis that each of these phosphoryl substitution restores full activity to the highly impaired A756G transfer reactions is subject to general acid-base catalysis. This ribozyme, and we conclude that the nucleobase of A756 is the mechanism requires a general base to deprotonate the attacking probable general acid in the cleavage reaction. nucleophile, and a general acid to protonate the oxyanion leaving group (Fig. 1). Results The most common chemical entities implicated in RNA Kinetic Analysis of Cleavage Using Modified VS Ribozyme and catalysis by the nucleolytic ribozymes are the nucleobases (2). Substrate. The analysis of VS ribozyme cleavage reactions was Guanine appears to play a catalytic role in the hairpin, hammer- performed using a ribozyme comprising helices II to VI acting head, GlmS, and Varkud satellite (VS) ribozymes, adenine in the in trans upon a substrate stem loop (Fig. 1A). The ribozyme hairpin, and VS and cytosine in the hepatitis delta virus (HDV) was prepared by transcription from a DNA template, and the BIOCHEMISTRY ribozyme. Crystal structures of the hairpin ribozyme (3) reveal substrate by a combination of chemical synthesis and sequential the presence of guanine (G8) and adenine (A38) bases juxta- ligations (Figs. S1 and S2). A 5′-PS-substituted scissile phosphate posed with the 2′-O and 5′-O, respectively, of the scissile phos- was introduced by synthesis of a GpA dinucleotide containing a phate, where they seem poised to act in general acid-base 5′ sulfur on the adenosine with o-nitrobenzyl protection on the catalysis. This is consistent with the pH dependence of the reac- tion (4) and its variation with functional group modifications (5–8). Author contributions: T.J.W., N.-S.L., J.L., J.K.F., J.A.P., and D.M.J.L. designed research; CHEMISTRY T.J.W., N.-S.L., J.L., and J.K.F. performed research; T.J.W. and D.M.J.L. analyzed data; In its simplest active form, the VS ribozyme comprises five and T.J.W., J.A.P., and D.M.J.L. wrote the paper. helices (II through VI) organized by two three-way junctions, The authors declare no conflict of interest. which acts in trans upon a substrate stem loop (helix I) with an This article is a PNAS Direct Submission. internal loop that contains the scissile phosphate (Fig. 1). The loop – 1To whom correspondence may be addressed. E-mail: [email protected] or jpicciri@ also contains the critical G638 (9). A756 (10 13) is contained chicago.edu. within an internal loop in helix VI. While no crystal structure 2Present address: Department of Pathology and Laboratory Medicine, The University of of the VS ribozyme has yet been solved, a small-angle X-ray scat- Rochester Medical Center, 601 Elmwood Avenue, Box 626, Rochester, NY 14642. tering-derived model places G638 and A756 in proximity to the This article contains supporting information online at www.pnas.org/lookup/suppl/ scissile phosphate (14). doi:10.1073/pnas.1004255107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1004255107 PNAS ∣ June 29, 2010 ∣ vol. 107 ∣ no. 26 ∣ 11751–11756 Downloaded by guest on September 24, 2021 A C G IV V U U C U U G C U G A A A U U G C G U A G C A G U G A C G U A A C U U U A A C G U A U U G U C A C I G C G C U G C U U C G U G G C C G U A A C 638 A U 621 A or DAP 5‘ III A G C G G G A U A C 3‘ C C G G G A 730 G U A A A A A A G U G U A A G C G G G G C U G UG C G G U A U U G G C U 5‘ GUGUCGCAAUC A C G U U C G C C C C G A CC A G U U A U G A C U G 3‘ U A A G A G G A A C A G A C A or II 756 VI G B C D O Gua N NH2 O NH N HO N N N NH2 O O H :B O HO N N NH P 2 O O O O oNO2Bn NH2 A H O O P HO OH Ade O N O O N S 2, 6-diaminopurine N N O OH O HO OH Fig. 1. The sequence of the VS ribozyme, the proposed mechanism, and the chemical structure of the 5′-phosphorothiolate substitution. (A). The sequence of the trans-acting ribozyme and substrate used here. The cleaved bond is arrowed. (B). Mechanism of general acid-base catalysis for the cleavage reaction of the VS ribozyme. (C). The chemical structure of a protected GpA dinucleotide with a 5′-phosphorothiolate linkage. oNO2Bn ¼ o-nitrobenzyl. (D). The chemical structure of 2,6-diaminopurine nucleoside. 2′-hydroxyl of the guanosine to prevent premature activation of the labile P-S bond during deprotection. Addition of the natural- the nucleophile (Fig. 1C). sequence (A756) ribozyme resulted in significant cleavage of both 0 32 The cleavage of radioactively ½5 - P-labeled substrate was 5′-PO and 5′-PS substrates. The 5′-PO substrate is poorly cleaved studied under single-turnover conditions immediately following by a ribozyme carrying the A756G substitution, consistent with deprotection of the 2′-hydroxyl nucleophile using ultraviolet previous observations (10). By contrast, the 5′-PS substrate was irradiation. Products of ribozyme cleavage were separated by substantially cleaved by the A756G ribozyme.
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