Complexity in orchestration of chemical groups near different cleavage sites in RNase P RNA mediated cleavage.
RNase P mediated cleavage of the tRNA(His) precursor does not rely on the formation of the "+73/294 interaction" to give the correct cleavage product, i.e. cleavage at -1, while other tRNA precursors that are cleaved at the canonical site +1 do. A previous model, here referred to as the "2'OH-model", predicts that the 2'OH at the canonical cleavage site would affect cleavage at -1. Here we used model RNA hairpin substrates mimicking the structural architecture of the tRNA(His) precursor cleavage site to investigate the role of 2'OH with respect to ground state binding and rate of cleavage in the presence and absence of the +73/294 interaction. Our data emphasize the importance of the 2'OH in the immediate vicinity of the scissile bond. Moreover, introduction of 2'H at the cleavage site did not affect cleavage at an alternative cleavage site to any significant extent. Our findings are therefore inconsistent with the 2'OH model. We favor a model where the 2'OH at the cleavage site influence Mg2+ binding in its vicinity, however we do not exclude the possibility that the 2'OH at the cleavage site interacts with RNase P RNA. Studying the importance of the 2'OH at different cleavage sites also indicated a higher dependence on the 2'OH at the cleavage site in the absence of the +73/294 interaction than in its presence. Finally, we provide data suggesting that N3 of U at position -1 in the substrate is most likely not involved in an interaction with RNase P RNA.
Brännvall
Mathias
M
Department of Cell and Molecular Biology, Box 596, Biomedical Centre, SE-751 24 Uppsala, Sweden.
Kirsebom
Leif A
LA
D001412:Bacillus subtilis / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D006639:Histidine / Q000737:chemistry; D007477:Ions; D007700:Kinetics; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry; D043262:Ribonuclease P / Q000737:chemistry*; D013997:Time Factors
D007477:Ions; D012329:RNA, Bacterial; D006639:Histidine; D012313:RNA; D012343:RNA, Transfer; D043262:Ribonuclease P