Reaction in vitro of some mutants of RNase P with wild-type and temperature-sensitive substrates.
The reaction of wild-type and two mutant derivatives of RNase P have been examined with wild-type and mutant substrates. We show that a mutant derivative of tRNA(Tyr)Su3, tRNA(Tyr)Su3A15, in which the G15.C48(57) base-pair essential for folding of the tRNA moiety is altered, is a temperature-sensitive suppressor in vivo. The precursor to tRNA(Tyr)Su3A15 is cleaved in a temperature-sensitive manner in vitro by RNase P and with a higher Km compared to the precursor to tRNA(Tyr)Su3. The precursor to tRNA(Tyr)Su3A2, another temperature-sensitive suppressor in vivo in which the G2.C71(80) base-pair in the acceptor stem is changed to A2.C71(80), behaves like the precursor to tRNA(Tyr)Su3 in vitro; that is, it is not cleaved in a temperature-sensitive manner. Therefore, there are at least two ways in which a suppressor tRNA can acquire a temperature-sensitive phenotype in vivo. One of the mutant derivatives of RNase P we have tested, rnpA49, which affects the protein cofactor of the enzyme, has a decreased kcat compared to wild-type, which can explain its phenotype in vivo.
Kirsebom
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LA
Department of Biology, Yale University, New Haven, CT 06520.
Altman
S
S
D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D009154:Mutation; D012329:RNA, Bacterial / Q000378:metabolism*; D012365:RNA, Transfer, Tyr / Q000378:metabolism; D043262:Ribonuclease P; D013696:Temperature
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012365:RNA, Transfer, Tyr; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli