Gel retardation analysis of E. coli M1 RNA-tRNA complexes.
We have analyzed complexes between tRNA and E. coli M1 RNA by electrophoresis in non-denaturing polyacrylamide gels. The RNA subunit of E. coli RNase P formed a specific complex with mature tRNA molecules. A derivative of the tRNA(Gly), endowed with the intron of yeast tRNA(ile) (60 nt), was employed to improve separation of complexed and unbound M1 RNA. Binding assays with tRNA(Gly) and intron-tRNA(Gly) as well as analysis of intron-tRNA/M1 RNA complexes on denaturing gels showed that one tRNA is bound per molecule of M1 RNA. A tRNA carrying a truncation as small as the 5'-nucleotide had a strongly reduced affinity to M1 RNA and was also a weak competitor in the cleavage reaction, suggesting that nucleotide +1 is a major determinant of tRNA recognition and that the thermodynamically stable tRNA-M1 RNA complex is relevant for enzyme function. Binding was shown to be dependent on the M1 RNA concentration in a cooperative fashion. Only a fraction of M1 RNAs (50-60%) readily formed a complex with intron-tRNA(Gly), indicating that distinct conformational subpopulations of M1 RNA may exist. Formation of the M1 RNA-tRNA(Gly), complex was very similar at 100 mM Mg++ and Ca++, corroborating earlier data that Ca++ is competent in promoting M1 RNA folding and tRNA binding. Determination of apparent equilibrium constants (app Kd) for tRNA(Gly) as a function of the Mg++ concentration supports an uptake of at least two additional Mg++ ions upon complex formation. At 20-30 mM Mg++, highest cleavage rates but strongly reduced complex formation were observed. This indicates that tight binding of the tRNA to the catalytic RNA at higher magnesium concentrations retards product release and therefore substrate turnover.
Hardt
W D
WD
Institut für Biochemie, Freie Universität Berlin, Germany.
Schlegl
J
J
Erdmann
V A
VA
Hartmann
R K
RK
D001483:Base Sequence; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D004722:Endoribonucleases / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D007438:Introns; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry; D012343:RNA, Transfer / Q000378:metabolism*; D012353:RNA, Transfer, Gly / Q000378:metabolism; D012355:RNA, Transfer, Ile / Q000378:metabolism; D043262:Ribonuclease P
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012353:RNA, Transfer, Gly; D012355:RNA, Transfer, Ile; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D008274:Magnesium; D002118:Calcium