title: Mechanistic characterization of the HDV genomic ribozyme: the cleavage site base pair plays a structural role in facilitating catalysis.
abstract: 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.
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Cerrone-Szakal
authors.forename: Durga M
authors.initials: DM
authors.lastname: Chadalavada
authors.forename: Barbara L
authors.initials: BL
authors.lastname: Golden
authors.forename: Philip C
authors.initials: PC
authors.lastname: Bevilacqua
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D002384:Catalysis; D016679:Genome, Viral; D003698:Hepatitis Delta Virus / Q000201:enzymology* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D016337:RNA, Catalytic; D012367:RNA, Viral