Gap-scan deletion analysis of Bacillus subtilis RNase P RNA.
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.
Waugh
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Department of Biology, Indiana University, Bloomington 47405.
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D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012349:RNA, Transfer, Asp / Q000378:metabolism; D043262:Ribonuclease P; D014158:Transcription, Genetic
D009841:Oligonucleotides; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012349:RNA, Transfer, Asp; D004722:Endoribonucleases; D043262:Ribonuclease P