General Acid–Base Catalysis Mediated by Nucleobases in the Hairpin Ribozyme

Stephanie Kath-Schorr, Timothy J. Wilson, Nan Sheng Li, Jun Lu, Joseph A. Piccirilli, David M. J. Lilley

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    72 Citations (Scopus)
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    The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of the phosphodiester backbone of RNA has been incompletely understood. There is experimental evidence for an important role for an adenine (A38) and a guanine (G8), and it has been proposed that these act in general acid-base catalysis. In this work we show that a large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 5′-oxygen atom at the scissile phosphate by sulfur (5′-PS), which is a much better leaving group. This is consistent with A38 acting as the general acid in the unmodified ribozyme. The rate of cleavage of the 5′-PS substrate by the A38P ribozyme increases with pH log-linearly, indicative of a requirement for a deprotonated base with a relatively high pKa. On substitution of G8 by diaminopurine, the 5′-PS substrate cleavage rate at first increases with pH and then remains at a plateau, exhibiting an apparent pKa consistent with this nucleotide acting in general base catalysis. Alternative explanations for the pH dependence of hairpin ribozyme reactivity are discussed, from which we conclude that general acid-base catalysis by A38 and G8 is the simplest and most probable explanation consistent with all the experimental data.

    Original languageEnglish
    Pages (from-to)16717-16724
    Number of pages8
    JournalJournal of the American Chemical Society
    Issue number40
    Early online date7 Sept 2012
    Publication statusPublished - 10 Oct 2012


    • RNA catalysis
    • catalytic mechanism
    • 5′-phosphorothiolate substitution

    ASJC Scopus subject areas

    • Catalysis
    • General Chemistry
    • Biochemistry
    • Colloid and Surface Chemistry


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