Manganese ions induce miscleavage in the Escherichia coli RNase P RNA-catalyzed reaction.
Cleavage by the endoribonuclease RNase P requires the presence of divalent metal ions, of which Mg2+ promotes most efficient cleavage. Here we have studied the importance of there being Mg2+ in RNase P RNA catalysis. It is demonstrated that addition of Mn2+ resulted in a shift of the cleavage site and that this shift was associated with a change in the kinetic constants, in particular kcat. Our data further suggest that the influence of Mn2+ on cleavage site recognition depends on the -1/+73 base-pair in the substrate and the +73/294 base-pair in the RNase P RNA-substrate (RS)-complex. Based on our data we suggest that cleavage in the presence of Mg2+ as the only divalent metal ion proceeds through an intermediate which involves the establishment of the +73/294 base-pair in the RS-complex. By contrast, addition of Mn2+ favours an alternative pathway which results in a shift of the cleavage site. We also studied the influence of Mn2+ on cleavage site recognition and the kinetics of cleavage using various RNase P RNA derivatives carrying substitutions in the region of RNase P RNA that base-pair with the 3' terminal end of the substrate. From these results we conclude that a change in the structure of this RNase P RNA domain influences the involvement of a divalent metal ion(s) in the chemistry of cleavage.
Brännvall
M
M
Department of Cell and Molecular Biology, Uppsala University, Uppsala, SE-751 24, Sweden.
Kirsebom
L A
LA
D020029:Base Pairing; D001483:Base Sequence; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D007854:Lead / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D043262:Ribonuclease P
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D007854:Lead; D008345:Manganese; D012313:RNA; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D008274:Magnesium