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

Timothy J. Wilson, Nan-Sheng Li, Jun Lu, John K. Frederiksen, Joseph A. Piccirilli (Lead / Corresponding author), David M. J. Lilley (Lead / Corresponding author)

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    Abstract

    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) = 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 phosphorothiolate-modified 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.

    Original languageEnglish
    Pages (from-to)11751-11756
    Number of pages6
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume107
    Issue number26
    DOIs
    Publication statusPublished - 29 Jun 2010

    Keywords

    • 5 '-phosphorothiolate
    • RNA catalysis
    • nucleolytic ribozymes
    • catalytic mechanism
    • DELTA VIRUS RIBOZYME
    • HAIRPIN RIBOZYME
    • ACTIVE-SITE
    • CRYSTAL-STRUCTURE
    • RNA CATALYSIS
    • MECHANISM
    • RESCUE
    • LOOP
    • GUANINE

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