Aminoacyl-tRNA-elongation factor Tu-ribosome interaction leading to hydrolysis of guanosine 5'-triphosphate

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Abstract
We investigated the elongation factor Tu (EF-Tu) dependent binding of Phe-tRNA and Phe-tRNAs with the nicks at positions 46, 37, and 17 to the Escherichia coli 70S ribosome-poly(U)-tRNAPhe complex. Binding of Phe-tRNA1-45+47-76, Phe-tRNA1-36-38-77, or Phe-tRNA1-16+17-76 to the 70S ribosome has been found to be poly(U).cntdot.tRNA dependent and, similar to that of intact Phe-tRNA, is inhibited by the antibiotic thiostrepton. We have further found that, contrart to a previous report [Modolell, J., Cabrer, B., Parmeggiani, A., and Vazquez, D. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 1796], the EF-Tu-ribosome GTPase mediated by Phe-tRNA is not inhibited by thiostrepton; rather, the drug stimulates the endogenous GTPase of the EF-Tu.cntdot.70S ribosome. Phe-tRNA fragments 47-76, 38-76, and 17-76 all promote the EF-Tu.cntdot.GTPase reaction in the presence of 70S .**GRAPHIC**. Moreover, since the GTPase-promoting activity of both the short and long fragments are similar, it appears that the most important aminoacyl transfer ribonucleic acid (aa-tRNA) interaction with EF-Tu occurs alongside its 3'' quarter. Thiostrepton slightly stimulates the GTPase activity of these Phe-tRNA fragments. Although the Phe-tRNA1-36+38-76 cannot bind to poly(U) during its binding to 70S ribosomes, its binding at high Mg2+ concentration occurs at the A site. Thus, most of the bound modified Phe-tRNA functions as the acceptor in the peptidyltransferase reaction. We interpret these results to mean that the GTP hydrolysis is triggered upon the initial contact of the ternary aa-tRNA.cntdot.EF-Tu.cntdot.GTP complex with a ribonsomal domain which may not be identical with the A site at which thiostrepton and EF-G act. The aa-tRNA molecule can reach the A site only after hydrolysis of GTP and removal of EF-Tu.cntdot.GDP from ribosomes.

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