Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B.subtilis RNase P protein

The bacterial RNase P holoenzyme catalyzes the formation of the mature 5'-e nd of tRNAs and is composed of an RNA and a protein subunit, Among the two folding domains of the RNase P RNA, the catalytic domain (C-domain) contain s the active site of this ribozyme, We investigated specific binding of the Bacillus subtilis C-domain with the B.subtilis RNase P protein and examine d the catalytic activity of this C-domain-P protein complex. The C-domain f orms a specific complex with the P protein with a binding constant of simil ar to0.1 muM. The C-domain-P protein complex and the holoenzyme are equally efficient in cleaving single-stranded RNA (similar to0.9 min(-1) at pH 7.8 ) and substrates with a hairpin-loop 3' to the cleavage site (similar to 40 min(-1)). The holoenzyme reaction is much more efficient with a pre-tRNA s ubstrate, binding at least 100-fold better and cleaving 10-500 times more e fficiently. These results demonstrate that the RNase P holoenzyme is functi onally constructed in three parts. The catalytic domain alone contains the active site, but has little specificity and affinity for most substrates. T he specificity and affinity for the substrate is generated by either the sp ecificity domain of RNase P RNA binding to a T stem-loop-like hairpin or RN ase P protein binding to a single-stranded RNA. This modular construction m ay be exploited to obtain RNase P-based ribonucleoprotein complexes with al tered substrate specificity.