A guanine nucleobase important for catalysis by the VS ribozyme

Wilson, Timothy J.; McLeod, Aileen; Lilley, David M.J.

doi:10.1038/sj.emboj.7601698

Cited by 87 publications

(149 citation statements)

References 44 publications

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“…The pH dependence of the cleavage rate for the natural VS ribozyme reaction is bell shaped, corresponding to the ionization of bases with apparent pK a values of 5.2 and 8.4, assigned to A756 and G638, respectively (9). In both cases the local environment shifts the pK a from the solution values of 3.8 for free adenosine and 9.4 for free guanosine.…”

Section: ′-Ps Substitution Restores Cleavage By a Ribozyme With A A756gmentioning

confidence: 92%

“…Replacement of G638 by diaminopurine (DAP, Fig. 1D) leads to significantly lower rates of substrate cleavage (9). Under standard conditions a 5′-PO substrate carrying the G638DAP substitution was cleaved by the A756 ribozyme at a rate of 0.037 min −1 .…”

Section: ′-Ps Substitution Restores Cleavage By a Ribozyme With A A756gmentioning

confidence: 99%

“…At high concentrations of Mg 2þ ions, the pH dependence of the cleavage reaction of the trans-acting VS ribozyme is bell shaped, fitting a model involving proton transfers in the transition state with participating groups of pK a ¼ 5.2 and 8.4 (9). Substitution of G638 by diaminopurine shifts the pH profile, corresponding to new pK a values of 4.6 and 5.6.…”

mentioning

confidence: 99%

“…1). The loop also contains the critical G638 (9). A756 (10-13) is contained within an internal loop in helix VI.…”

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confidence: 99%

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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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Section: ′-Ps Substitution Restores Cleavage By a Ribozyme With A A756gmentioning

confidence: 92%

Section: ′-Ps Substitution Restores Cleavage By a Ribozyme With A A756gmentioning

confidence: 99%

mentioning

confidence: 99%

“…1). The loop also contains the critical G638 (9). A756 (10-13) is contained within an internal loop in helix VI.…”

mentioning

confidence: 99%

See 2 more Smart Citations

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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“…Purine bases are most prevalent, and the majority of ribozymes use a deprotonated guanine nucleobase as a general base in the cleavage reaction (16,19,(39)(40)(41)(42)(43)(44)(45).…”

Section: The Role Of Nucleobases In Ribozyme Catalysismentioning

confidence: 99%