Abstract
The negative strand of the satellite RNA of tobacco ringspot virus [(-) sTRSV] is a selfcleaving RNA, of which self-cleaving domain is called the hairpin ribozyme. The negative strand of the satellite RNA of arabis mosaic virus [(-) sArMV] has been suggested to have a hairpin ribozyme-like secondary structure, and we have previously shown that this hairpin domain of (-) sArMV has ribozyme activity. Here we report characterization of the cleavage reaction of the (-) sArMV hairpin ribozyme. Mutagenesis analyses in a trans-acting 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 reaction), however, the reaction of the RNA containing the wild-type sequence proceeds highly efficiently. This result suggests that the inefficient cleavage of the wild-type substrate in trans-acting system may be due to low efficiency at the substrate-binding step but not at the chemical cleavage step in the reaction. We also constructed a chimeric ribozyme between the catalytic hairpin domain from (-) sArMV and the substrate-binding site from (-) sTRSV. This chimeric ribozyme had the highest activity among the trans-acting hairpin ribozymes tested.
