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 Ml 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 Ml 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.