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

Abstract

Recent suggestions that elongation factor Tu (EF-Tu) is specific for 2'-0-aminoacyl-tRNA, as compared with the 3'-isomer, prompted us to assay [3Hlaminoacyl-tRNAs 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 aminaoc l-tRNAs to EFTuGTP was measured both as a function of the ability of EFTuGTP to diminish the rate of chemical deacylation of[3H]aminoacyl-tRNAs and by gel filtration of the individual ternary complexes. Fifteen different tRNA isoacceptors were tested by the deacylation procedure, including three (tRNAAsP, tRNACYs, and tRNATYr) for which isomeric modified aminoacyl-tRNAs were available. All of the modified aminoacyltRNAs were protected from deacylation, although generally to a lesser extent than the corresponding unmodified species.Six modified tRNA isoacceptors (including tRNATrP and tRNATYr, fQr which both modified aminoacyl-tRNAs were accessible by enzymatic aminoacylation) were used in gel filtration experiments to permit direct measurement of the individual aminoacyl-tRNA-EF-Tu-GTP complexes. These experiments were also done in the presence of equimolar amounts of the corresponding unmodified [14Claminoacyl-tRNAs, and the relative affinities for a limiting amount of EF-TuGTP were measured. The results were completely consistent with those obtained by the deacylation procedure and indicated that EF-Tu can bind to both positionalisomers of aminoacyl-tRNA with no

Identification of thermodynamically relevant interactions between EF-Tu and backbone elements of tRNA

“…Finally, a contact between the side-chain of Glu271 and the 2 H hydroxyl group at position 76 was proposed to be critical for maintaining the location of the amino acid residue on the 3 H hydroxyl group when bound to EF-Tu. This interaction appeared to settle a dispute as to which isomeric aminoacyl linkage was required for EF-Tu binding, as previous biochemical studies did not agree on the issue (Alford et al, 1979;Forster et al, 1994;Hecht et al, 1977;Taiji et al, 1985;Wagner et al, 1982). However, the current set of experiments, using a more quantitative assay than the previous studies, show that removal of the 2 H hydroxyl group at position 76 has no effect on the af®nity of Ala-YFA2 for EF-Tu (GTP).…”

Effector region of the translation elongation factor EF-Tu.GTP complex stabilizes an orthoester acid intermediate structure of aminoacyl-tRNA in a ternary complex.

“…By using the nonisomerizable 2'-and 3'-deoxy aminoacyl-tRNAs, respectively, it was demonstrated that both 2'-aminoacyl-and 3'-aminoacyl-tRNA interacted with the EF-Tu-GTP binary complex, although their affinity relative to native aminoacyl-tRNA was reduced (7,8). From fast kinetic experiments using native aminoacyl-tRNA, it was later confirmed that EF-Tu-GTP bound both isomers and acted as an isomerase to produce the 3' isomer, which is competent as a donor in the peptidyl transfer reaction (9,10).…”