Gap-scan deletion analysis of Bacillus subtilis RNase P RNA

Abstract

We carried out an exhaustive deletion analysis of Bacillus subtilis ribonuclease P (RNase P) RNA, seeking sequences that are essential for its catalytic activity. A collection of partially deleted RNase P RNA genes was used to construct templates for synthesis, by in vitro transcription, of circularly permuted RNA molecules that lack various wild-type sequences. The mutant RNAs were assayed for catalytic activity in vitro, using a precursor of B. subtilis tRNAAsp as a substrate. Gap-scan deletion analysis revealed that most of the RNase P RNA sequence is important for activity; only two substantive deletions did not dramatically inhibit pre-tRNA processing in vitro. One part of the molecule (nucleotides 225-270) seemed particularly sensitive to deletion, but considering a collection of mutants with overlapping deletion gaps, it was possible to remove every residue in the RNase P RNA without completely abolishing its catalytic activity. Thus, the catalytic mechanism of RNase P does not depend absolutely on a single, particular nucleotide or local sequence for activity.