GEL RETARDATION ANALYSIS OF ESCHERICHIA-COLI M1 RNA - TRANSFER-RNA COMPLEXES

We have analyzed complexes between tRNA and E.coli Ml RNA by electroph oresis in non-denaturing polyacrylamide gels. The RNA subunit of E. co li RNase P formed a specific complex with mature tRNA molecules. A der ivative of the tRNA(Gly), endowed with the intron of yeast tRNA(Ile) ( 60 nt), was employed to improve separation of complexed and unbound Ml RNA. Binding assays with tRNA(Gly) and intron-tRNA(Gly) as well as an alysis of intron-tRNA/M1 RNA complexes on denaturing gels showed that one tRNA is bound per molecule of Ml RNA. A tRNA carrying a truncation as small as the 5'-nucleotide had a strongly reduced affinity to Ml R NA and was also a weak competitor in the cleavage reaction, suggesting that nucleotide + 1 is a major determinant of tRNA recognition and th at the thermodynamically stable tRNA-Ml RNA complex is relevant for en zyme function. Binding was shown to be dependent on the Ml RNA concent ration in a cooperative fashion. Only a fraction of Ml RNAs (50-60%) r eadily formed a complex with intron-tRNA(Gly), indicating that distinc t conformational subpopulations of Ml RNA may exist. Formation of the Ml RNA-tRNA(Gly) complex was very similar at 100 mM Mg++ and Ca++, cor roborating earlier data that Ca++ is competent in promoting Ml RNA fol ding and tRNA binding. Determination of apparent equilibrium constants (app Kd) for tRNA(Gly) as a function of the Mg+ + concentration suppo rts an uptake of at least two additional Mg++ ions upon complex format ion. At 20 - 30 mM Mg++, highest cleavage rates but strongly reduced c omplex formation were observed. This indicates that tight binding of t he tRNA to the catalytic RNA at higher magnesium concentrations retard s product release and therefore substrate turnover.