Phosphate residues of antigenomic HDV ribozyme important for catalysis that are revealed by phosphorothioate modification

摘要

The aim of our study was to obtain new details on the role of phosphate residues in antigenomic hepatitis delta virus (HDV) ribozyme for the development of metal ion binding sites and their participation in the cleavage mechanism. In these studies, the wild-type ribozyme and four in vitro selected variants, R37, R20, R25 and R51, were used. The application of nucleotide phosphorothioates and the NAIM (nucleotide analog interference mapping) technique for the cis-acting ribozymes, wild-type, R25 and R51, revealed the importance of the J4/2 and P1.1 regions in the catalysis. Interestingly, in the wild-type ribozyme, the largest interference effects were observed close to catalytic C76 in the presence of Ca~(2+) , while in the case of Mg~(2+) were in the structurally important helix P1.1. The results obtained for R25 and R51 suggest different coordination of the divalent ions to the phosphate residues within the ribozyme catalytic core. Additionally, replacing the non-bridging oxygen atoms on sulfur in a phosphate group at the cleavage site in tans-acting ribozyme variants showed that interactions between pro-R_P and pro-S_P oxygen atoms, and catalytic metal ions, had moderate effects on the cleavage reaction. In the wild-type ribozyme, the ratio of S_P/R_P isomer cleavage rates decreased from 25 for Mg~(2+)-induced cleavage to ca. 4 when thiophilic Mn~(2+) or Cd~(2+) were added; thus a "rescue effect" was observed. Interestingly, the R37, R20, R25 and R51 ribozymes showed a reduced R_P/S_P ratio of cleavage rates and much smaller "rescue effects". This suggests that the binding of divalent metal ions in the vicinity of the phosphate group at the cleavage site is very sensitive to the overall ribozyme structure.