Requirement for canonical base pairing in the short pseudoknot structure of genomic hepatitis delta virus ribozyme

The tertiary structure of the 3'-cleaved product of the genomic hepatitis d elta virus (HDV) ribozyme was solved by X-ray crystallographic analysis, In this structure, three single-stranded regions (SSrA, -B and -C) interact i ntricately with one another via hydrogen bonds between nucleotide bases, ph osphate oxygens and 2'-OHs to form a nested double pseudoknot structure, Am ong these interactions, two Watson-Crick (W-C) base pairs, 726G-710C and 72 7G-709C, that form between SSrA and SSrC (P1,1) seem to be especially impor tant for compact folding. To characterize the importance of these base pair s, ribozymes were subjected to in vitro selection from a pool of RNA molecu les randomly substituted at positions 709, 710, 726 and 727, The results es tablish the importance of the two W-C base pairs for activity, although som e mutants are active with one G-C base pair, In addition, the kinetic param eters were analyzed in all 16 combinations with two canonical base pairs. C omparison of variant ribozymes with the wild-type ribozyme reveals that the difference in reaction rates for these variants (Delta Delta G(double dagg er)) is not simply accounted for by the differences in the stability of P1, 1 (Delta Delta G(37)(0)) The role played by Mg2+ ions in formation of the P 1,1 structure is also discussed.