Exploring the minimal substrate requirements for trans-cleavage by RNase Pholoenzymes from Escherichia coli and Bacillus subtilis

We analysed the processing of small bipartite model substrates by Escherich ia coli and Bacillus subtilis RNase P and corresponding hybrid enzymes. We demonstrate specific trans-cleavage of a model substrate with a 4 bp stem a nd a 1 nucleotide (nt) 5' flank, representing to date the smallest mimic of a natural RNase P substrate that could be processed in trans at the canoni cal RNase P cleavage site. Processing efficiencies decreased up to 5000-fol d when the 5' flank was shortened from 3 to 1 nt. Reduction of the 5' flank to 1 nt was more deleterious than reducing the stem from 7 to 4 bp, althou gh the 4 bp duplex formed only transiently, in contrast to the stable 7 bp duplex, These results indicate that the crucial contribution of nt -2 in th e single-stranded 5' flank to productive interaction is a general feature o f A- and B-type bacterial RNase P enzymes, We also showed that an Rp-phosph orothioate modification at nt -2 interferes with processing, Bacterial RNas e P holoenzymes are also capable of cleaving single-stranded RNA oligonucle otides as short as 5 nt, yielding RNase P-specific 5'-phosphate and 3'-OH t ermini, with measured turnover rates of up to 0.7 min(-1). All cleavage sit es were at least 2 nt away from the 5' and 3' ends of the oligonucleotides. Some cleavage site preferences were observed dependent on the identity of the RNase P RNA subunit.