Differential modulation of the elongation-factor-G GTPase activity by tRNA bound to the ribosomal A-site or P-site.

Abstract

The role of tRNA in the regulation of the GTPase activity dependent on the ribosome elongation factor G (EF-G), uncoupled from polypeptide synthesis, was studied in a highly purified system with well-characterized NH4Cl-washed ribosomes. In the range of physiological concentration of monovalent cations, the nonenzymatic binding of tRNAPhe, Phe-tRNAPhe and AcPhe-tRNAPhe to the ribosome · poly(U) complex was found either to stimulate or to inhibit the EF-G GTPase activity depending on whether the ribosomal P-site or both ribosomal A and P-siteswere respectively occupied. In both cases the Km of the GTPase reaction was not modified, indicating that bound tRNA has no direct effect on the catalytic center of the reaction. The stimulation of the EF-G GTPase by tRNA bound to the P-site varies with EF-G concentration; at equimolar ratio of EF-G to ribosomes, the ribosomes carrying tRNA are three or four times as active as tRNA-free ribosomes, but both ribosomal populations show the same ability to trigger the reaction at infinite EF-G concentration. Since the release of the EF-G · GDP complex is the rate-limiting step of the uncoupled EF-G GTPase with tRNA-free ribosomes, the tRNAPhe bound to the P-site stimulates the turnover of EF-G GTPase, probably by promoting a faster release of EF-G · GDP complex from the ribosome. By contrast, the additional occupation of the A-site by tRNA causes an inhibition of the activity, which is independent of the EF-G concentration and appears to be a consequence of the inability of the ribosome to trigger the reaction. Poly(U)-directed poly(phenyla1anine) synthesis was found to require ionic conditions similar to those needed for the stimulation of the EF-G GTPase by tRNA, emphasizing the physiological significance of this phenomenon. From our results, the ribosomal activity in the EF-G GTPase uncoupled from polypeptide synthesis appears to be controlled by tRNA through mechanisms which are also implicated in the regulation of the coupled reaction [Chinali, G. and Parmeggiani, A. (1980) J. Biol. Chem. 255, 7455–7459].