A physical assay for and kinetic analysis of the interactions between M1 RNA and tRNA precursor substrates.
A gel-shift assay was devised to detect stable enzyme-substrate (E-S) complexes between M1 RNA, the catalytic subunit of RNase P from Escherichia coli, and its tRNA precursor substrates. The use of deletion derivatives of M1 RNA in the gel-shift assay has allowed us to identify regions of the enzyme that are involved in the binding of the substrate or that are necessary for catalytic activity. Fragments of substrates that contain the 3' CCA sequence bind preferentially to regions in the 5' half of M1 RNA, while 5' leader sequences interact primarily with regions in the 3' half of M1 RNA. The 5' leader sequence present in the precursor to tRNA(Tyr)su3 from E. coli plays an important role in the formation of stable E-S complexes with M1 RNA. The CCA sequence at the 3' end of precursor tRNA substrates is involved in the product-release step of the reaction that is catalyzed by M1 RNA. Direct measurements of the concentrations of all the components in the reaction catalyzed by M1 RNA facilitated a new approach to the kinetic analysis of the action of the enzyme.
Guerrier-Takada
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Department of Biology, Yale University, New Haven, Connecticut 06520.
Altman
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D001483:Base Sequence; D004587:Electrophoresis, Agar Gel; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000378:metabolism*; D012329:RNA, Bacterial / Q000187:drug effects / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D043262:Ribonuclease P; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D008670:Metals; D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli