PMID:11454206
| Citation |
Hansen, A, Pfeiffer, T, Zuleeg, T, Limmer, S, Ciesiolka, J, Feltens, R and Hartmann, RK (2001) Exploring the minimal substrate requirements for trans-cleavage by RNase P holoenzymes from Escherichia coli and Bacillus subtilis. Mol. Microbiol. 41:131-43 |
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| Abstract |
We analysed the processing of small bipartite model substrates by Escherichia coli and Bacillus subtilis RNase P and corresponding hybrid enzymes. We demonstrate specific trans-cleavage of a model substrate with a 4 bp stem and 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 canonical RNase P cleavage site. Processing efficiencies decreased up to 5000-fold 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, although 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 the single-stranded 5' flank to productive interaction is a general feature of A- and B-type bacterial RNase P enzymes. We also showed that an Rp-phosphorothioate modification at nt -2 interferes with processing. Bacterial RNase P holoenzymes are also capable of cleaving single-stranded RNA oligonucleotides as short as 5 nt, yielding RNase P-specific 5'-phosphate and 3'-OH termini, with measured turnover rates of up to 0.7 min-1. All cleavage sites 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. |
| Links | |
| Keywords |
Bacillus subtilis/enzymology; Endoribonucleases/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins; Holoenzymes/metabolism; Kinetics; Oligonucleotides/metabolism; RNA, Bacterial/chemistry; RNA, Bacterial/metabolism; RNA, Catalytic/metabolism; Ribonuclease P; Substrate Specificity |
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