Mechanistic characterization of the HDV genomic ribozyme: the cleavage site base pair plays a structural role in facilitating 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(+)*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 Mg(2+)-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(+)*C. Rates for these native-folding ribozymes displayed highly similar pH and Mg(2+) concentration dependencies, with the exception of the A(+)*C ribozyme, which deviated at high pH. None of the four ribozymes underwent miscleavage. These observations support the A(+)*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.