Mechanistic characterization of the HDV genomic ribozyme: The cleavage site base pair plays a structural role in facilitating catalysis

作者：Cerrone Szaka Andrea L; Chadalavada Durga M; Colden Barbara L; Bevilacqua Philip C^*

来源：RNA-A Publication of the RNA Society, 2008, 14(9): 1746-1760.

DOI：10.1261/rna.1140308

• ribozyme
• RNA folding
• GU wobble
• catalysis

The hepatitis delta virus (HDV) ribozyme occurs in the genomic and antigenomic strands of the HDV RNA and within mammalian transcriptomes. Previous kinetic studies suggested that a wobble pair (G-U or A(+)center dot C) is preferred at the cleavage site; however, the reasons for this are unclear. We conducted sequence comparisons, which indicated that while G-U is the most prevalent combination at the cleavage site, G-C occurs to a significant extent in genomic HDV isolates, and G-U, G-C, and A-U pairs are present in mammalian ribozymes. We analyzed the folding of genomic HDV ribozymes by free energy minimization and found that variants with purine-pyrimidine combinations at the cleavage site are predicted to form native structures while pyrimidine-purine combinations misfold, consistent with earlier kinetic data and sequence comparisons. To test whether the cleavage site base pair contributes to catalysis, we characterized the pH and Mg2+-dependence of reaction kinetics of fast-folding genomic HDV ribozymes with cleavage site base pair purine-pyrimidine combinations: G-U, A-U, G-C, and A(+)center dot C. Rates for these native-folding ribozymes displayed highly similar pH and Mg2+ concentration dependencies, with the exception of the A(+)center dot C ribozyme, which deviated at high pH. None of the four ribozymes underwent miscleavage. These observations support the A(+)center dot C ribozyme as being more active with a wobble pair at the cleavage site than with no base pair at all. Overall, the data support a model in which the cleavage site base pair provides a structural role in catalysis and does not need to be a wobble pair.

• 出版日期2008-9