The 3' substrate determinants for the catalytic efficiency of the Bacillus subtilis RNase P holoenzyme suggest autolytic processing of the RNase P RNA in vivo.
We investigated the catalytic efficiency and the specificity of the Bacillus subtilis RNase P holoenzyme reaction with substrates that contain a single strand, a hairpin loop, or a tRNA 3' to the cleavage site. At a saturating ribozyme concentration, RNase P can cleave a single-stranded RNA at approximately 0.6 min(-1) at pH 7.8. Replacing the single-stranded RNA 3' to the cleavage site by a hairpin loop or by the yeast tRNA(Phe) increases the cleavage rate by up to approximately 600-fold and approximately 3,200-fold, respectively. These results show that compared to a single-stranded RNA substrate, the cleavage rate for the holoenzyme reaction is primarily enhanced by an acceptor-stem-like helix. Substrate binding, approximately 7-10 microM for a single-stranded RNA, improves by approximately 1,000-fold upon the addition of the tRNA. The efficiency of the RNase P holoenzyme cleaving a single-stranded RNA is sufficiently high to consider autolytic processing of the RNase P RNA (denoted P RNA) transcript in the cell. The addition of the RNase P protein to a precursor form of the P RNA in vitro results in autolytic processing of the 5' and the 3' end of this precursor in a matter of minutes. Autolytic processing produces the reported 5' end of the mature P RNA. The precise 3' end generated by autolytic processing is different over the course of the reaction and the final product is 4 nt shorter than the reported 3' end of the B. subtilis P RNA. The observed 3' end in vitro is consistent with the property of the holoenzyme reaction with single-stranded RNA substrates. The discrepancy with the reported 3' end may be due to other processing events in vivo or inaccurate determination of the mature 3' end of the P RNA isolated from the cell. We propose that the mature B. subtilis P RNA is generated at least in part by autolytic processing upon the binding of the RNase P protein to the precursor P RNA.
Loria
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A
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637, USA.
Pan
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T
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002384:Catalysis; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000235:genetics; D043262:Ribonuclease P; D012261:Ribonucleoproteins / Q000378:metabolism; D013379:Substrate Specificity; D015003:Yeasts / Q000235:genetics
D020035:Holoenzymes; D012322:RNA Precursors; D016337:RNA, Catalytic; D012360:RNA, Transfer, Phe; C039919:RNA, recombinant; D012261:Ribonucleoproteins; D012313:RNA; D004722:Endoribonucleases; D043262:Ribonuclease P