Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site.
Like the translational elongation factor EF-Tu, RNase P interacts with a large number of substrates where RNase P with its RNA subunit generates tRNAs with matured 5' termini by cleaving tRNA precursors immediately 5' of the residue at +1, i.e. at the position that corresponds to the first residue in tRNA. Most tRNAs carry a G+1C+72 base pair at the end of the aminoacyl acceptor-stem whereas in tRNA(Gln) G+1C+72 is replaced with U+1A+72. Here, we investigated RNase P RNA-mediated cleavage as a function of having G+1C+72 versus U+1A+72 in various substrate backgrounds, two full-size tRNA precursors (pre-tRNA(Gln) and pre-tRNA(Tyr)Su3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G+1C+72 with U+1A+72 influenced ground state binding, cleavage efficiency under multiple and single turnover conditions in a substrate-dependent manner. Interestingly, we observed differences both in ground state binding and rate of cleavage comparing two full-size tRNA precursors, pre-tRNA(Gln) and pre-tRNA(Tyr)Su3. These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step. In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA. Finally, we provide evidence that the structural architecture influences Mg2+ binding, most likely in its vicinity.
Kikovska
Ema
E
Department of Cell and Molecular Biology Box 596, Biomedical Centre, SE-751 24 Uppsala, Sweden.
Brännvall
Mathias
M
Kufel
Joanna
J
Kirsebom
Leif A
LA
D020029:Base Pairing; D001483:Base Sequence; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism*; D012351:RNA, Transfer, Gln / Q000737:chemistry / Q000378:metabolism; D043262:Ribonuclease P / Q000378:metabolism*; D013379:Substrate Specificity
D012322:RNA Precursors; D012351:RNA, Transfer, Gln; D012343:RNA, Transfer; D043262:Ribonuclease P; D008274:Magnesium