Fluorescence characterization of the interaction of various transfer RNA species with elongation factor Tu.cntdot.GTP: evidence for a new functional role for elongation factor Tu in protein biosynthesis

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Abstract
The ubiquity of elongation factor Tu (EF-Tu)-dependent conformational changes in aminoacyl-tRNA (aa-tRNA) and the origin of the binding energy associated with aa-tRNA.cntdot.EF-Tu.cntdot.GTP ternary complex formation have been examined spectroscopically. Fluorescein was attached covalently to the 4-thiouridine base at position 8 (s4U-8) in each of four elongator tRNAs (Ala, Met-m, Phe, and Val). Although the probes were chemically identical, their emission intensities in the free aa-tRNAs differed by nearly 3-fold, indicating that the dyes were in different environments and hence that the aa-tRNAs had different tertiary structures near s4U-B. Upon association with EF-Tu.cntdot.GTP, the emission intensities increased by 244%, 57%, or 15% for three aa-tRNAs due to a change in tRNA conformation; the fourth aa-tRNA exhibited no fluorescence change upon binding to EF-Tu.cntdot.GTP. Despite the great differences in the emission intensities of the free aa-tRNAs and in the magnitudes of their EF-Tu-dependent intensity increases, the emission intensity per aa-tRNA molecule was nearly the same (within 9% of the average) for the four aa-tRNAs when bound to EF-Tu.cntdot.GTP. Thus, the binding of EF-Tu.cntdot.GTP induced or selected a tRNA conformation near s4U-8 that was very similar, and possibly the same, for each aa-tRNA species. It therefore appears that EF-Tu functions, at least in part, by minimizing the conformational diversity in aa-tRNAs prior to their beginning the recognition and binding process at the single decoding site on the ribosome. Since an EF-Tu-dependent fluorescence change was also observed with fluorescein-labeled tRNAPhe, the protein-dependent structural change is effected by direct interactions between EF-Tu and the tRNA and does not require the aminoacyl group. The Kd of the tRNAPhe.cntdot.EF-Tu.cntdot.GTP ternary compelx was determined, at equiibrium, to be 2.6 .mu.M by the ability of the unacylated tRNA to compete with fluorescent Phe-tRNA for binding to the protein. Comparison of this Kd with that of the Phe-tRNA ternary complex showed that in this case the aminoacyl moiety contributed 4.3 kcal/mol toward ternary complex formation at 6.degree. C but that the bulk of the binding energy in the ternary complex was derived from direct protein-tRNA interactions. The acetylation of Phe-tRNAPhe had a greater effect on its ternary complex Kd than did the formylation of Met-tRNAMet, which suggests that N-blocked aa-tRNA species are prevented sterically from filling the site on EF-Tu that binds the .alpha.-amino group. The affinity of EF-Tu.cntdot.GTP for both elongator and initiator Met-TRNAMet species has also been quantified.