Synthesis and self-cleavage reaction of a chimeric molecule between RNase P-RNA and its model substrate.

We synthesized a chimeric RNA between the catalytic RNA subunit of RNase P from Escherichia coli (M1 RNA) and a model substrate of the enzyme. The model substrate is the smallest substrate of RNase P, having a simple stem-loop structure. This model substrate was added to the 3'-end of M1 RNA. This chimeric molecule, which we call M1 RNA-MS, is a self-cleaving RNA and is cleaved much more efficiently than the M1 RNA-pre-tRNA, an artificial self-cleaving RNA previously synthesized [Kikuchi et al. (1993) Nucleic Acids Res. 21, 4685-4689], that consists of a full-size tRNA precursor and the M1 RNA. The self-cleavage of M1 RNA-MS at 10 mM Mg2+ was an intramolecular reaction (cis-cleavage). Ca2+ supported the self-cleavage of M1 RNA-MS as effectively as Mg2+, although the self-cleavage of M1 RNA-pre-tRNA proceeded with low efficiency in the presence of Ca2+ as the only metal ion. Future application of the M1 RNA-MS molecule to the in vitro evolution of the M1 RNA and other experiments is proposed.

Cis and trans reactions of hairpin ribozymes derived from the negative strand of arabis mosaic virus satellite RNA.

We have previously shown that the hairpin ribozyme-like structure of the negative strand of the satellite RNA of arabis mosaic virus [(- )sArMV] has indeed ribozyme activity. However, some mutagenesis analyses revealed surprisingly that the wild type ribozyme was less active than almost all the other mutant ribozymes tested. These results were derived from a trans-acting system. Here we tested this ribozyme activity in a cis-acting system. We show that the (-)s ArMV hairpin ribozyme has different target-site specificities between cis and trans cleavages. The wild type ribozyme has the highest self-cleaving activity among the ribozyme variants tested.