IN-VITRO ACTIVITY OF THE HAIRPIN RIBOZYME DERIVED FROM THE NEGATIVE STRAND OF ARABIS MOSAIC-VIRUS SATELLITE RNA

The negative strand of the satellite RNA of tobacco ringspot virus [(- )sTRSV] is a self-cleaving RNA, of which self-cleaving domain is calle d the hairpin ribozyme, The negative strand of the satellite RNA of ar abis mosaic virus [(-)sArMV] has been suggested to have a hairpin ribo zyme-like secondary structure, and we have previously shown that this hairpin domain of (-)sArMV has ribozyme activity, Here we report chara cterization of the cleavage reaction of the (-)sArMV hairpin ribozyme, Mutagenesis analyses in a transacting system revealed, surprisingly, that the wild-type ribozyme was less active than almost all the other mutant ribozymes tested, In a cis-acting system (self-cleaving reactio n), however, the reaction of the RNA containing the wild-type sequence proceeds highly efficiently, This result suggests that the inefficien t cleavage of the wild-type substrate in trans-acting system may be du e to low efficiency at the substrate-binding step but not at the chemi cal cleavage step in the reaction, We also constructed a chimeric ribo zyme between the catalytic hairpin domain from (-)sArMV and the substr ate-binding site from (-)sTRSV, This chimeric ribozyme had the highest activity among the traits-acting hairpin ribozymes tested.