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 Bacillu s subtilis RNase P holoenzyme reaction with substrates that contain a singl e strand, a hairpin loop, or a tRNA 3' to the cleavage site. At a saturatin g ribozyme concentration, RNase P can cleave a single-stranded RNA at simil ar to0.6 min(-1) at pH 7.3, Replacing the single-stranded RNA 3' to the cle avage site by a hairpin loop or by the yeast tRNA(Phe) increases the cleava ge rate by up to similar to 600-fold and similar to3,200-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 accep tor-stem-like helix. Substrate binding, similar to7-10 muM for a single-str anded RNA, improves by similar to1,000-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 (deno ted 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 th e 5' and the 3' end of this precursor in a matter of minutes. Autolytic pro cessing produces the reported 5' end of the mature P RNA. The precise 3' en d generated by autolytic processing is different over the course of the rea ction 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 pro perty of the holoenzyme reaction with single-stranded RNA substrates. The d iscrepancy with the reported 3' end may be due to other processing events i n vivo or inaccurate determination of the mature 3' end of the P RNA isolat ed 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 p rotein to the precursor P RNA.