Cleavage Mediated by the Catalytic Domain of Bacterial RNase P RNA
Like other RNA molecules RNase P RNA (RPR) is composed of domains and these have different functions. Here we provide data demonstrating that the catalytic (C) domain of Escherichia coli (Eco) RPR when separated from the specificity (S) domain mediates cleavage using various model RNA hairpin loop substrates. Compared to full-length Eco RPR the rate constant, kobs, of cleavage for the truncated RPR (CP RPR) was reduced 30- to 13000-fold depending on substrate. Specifically, the structural architecture of the − 1/+ 73 played a significant role where a C− 1/G+ 73 pair had the most dramatic effect on kobs. Substitution of A248 (E. coli numbering), positioned near the cleavage site in the RNase P-substrate complex, with G in the CP RPR resulted in 30-fold improvement in rate. In contrast, strengthening the interaction between the RPR and the 3' end of the substrate only had a modest effect. Interestingly, although deleting the S-domain gave a reduction in the rate it resulted in a less erroneous RPR with respect to cleavage site selection. These data support and extend our understanding of the coupling between the distal interaction between the S-domain and events at the active site. Our findings will also be discussed with respect to the structure of RNase P RNA derived from different organisms. View high quality image (228K) âº Deleting the S-domain lowers the rate 30- to 13000-fold depending on substrate. âº The lower rate depends on the structure of the -1/+73 base pair in the substrate. âº Mutations in the RNA catalyst partly compensate for the lower rate. âº Deleting the S-domain gives a less erronenous RNA catalyst.