Isomeric aminoacyl-tRNAs are both bound by elongation factor Tu.

Abstract

Recent suggestions that elongation factor Tu (EF-Tu) is specific for 2''-O-aminoacyl-tRNA, as compared with the 3''-isomer, prompted the assay of [3H]aminoacyl-tRNA from Escherichia coli terminating in 2''- or 3''-deoxyadenosine for binding to EF-Tu to determine the possible positional specificity of the factor. Binding of modified aminoacyl-tRNA to EF-Tu.cntdot.GTP was measured both as a function of the ability of EF-Tu.cntdot.GTP to diminish the rate of chemical deacylation of [3H]aminoacyl-tRNA and by gel filtration of the individual ternary complexes. Fifteen different tRNA isoacceptors were tested by the deacylation procedure, including 3 (tRNAAsp, tRNACys and tRNATyr) for which isomeric modified aminoacyl-tRNA were available. All of the modified aminoacyl-tRNA were protected from deacylation, although generally to a lesser extent than the corresponding unmodified species. Six modified tRNA isoacceptors (including tRNATrp and tRNATyr, for which both modified aminoacyl-tRNA were accessible by enzymatic aminoacylation) were used in gel filtration experiments to permit direct measurement of the individual aminoacyl-tRNA-EF-Tu.cntdot.GTP complexes. The same experiments were done in the presence of equimolar amounts of the corresponding unmodified [14C]aminoacyl-tRNA, and the relative affinities for a limiting amount of EF-Tu.cntdot.GTP were measured. The results were completely consistent with those obtained by the deacylation procedure and indicated that EF-Tu can bind to both positional isomers of aminoacyl-tRNA with no obvious preference for either.