The translocation of tRNA and mRNA through the ribosome is promoted by elongation factor G (EF-G), a GTPase that hydrolyzes GTP during the reaction. Recently, it was reported that, in contrast to previous observations, the affinity of EF-G was much weaker for GTP than for GDP and that ribosome-catalyzed GDP-GTP exchange would be required for translocation [Zavialov AV, Hauryliuk VV, Ehrenberg M (2005) J Biol 4:9]. We have reinvestigated GTP͞GDP binding and show that EF-G binds GTP and GDP with affinities in the 20 to 40 M range (37°C), in accordance with earlier reports. Furthermore, GDP exchange, which is extremely rapid on unbound EF-G, is retarded, rather than accelerated, on the ribosome, which, therefore, is not a nucleotide-exchange factor for EF-G. The EF-G⅐GDPNP complex, which is very labile, is stabilized 30,000-fold by binding to the ribosome. These findings, together with earlier kinetic results, reveal that EF-G enters the pretranslocation ribosome in the GTP-bound form and indicate that, upon ribosomecomplex formation, the nucleotide-binding pocket of EF-G is closed, presumably in conjunction with GTPase activation. GTP hydrolysis is required for rapid tRNA-mRNA movement, and Pi release induces further rearrangements of both EF-G and the ribosome that are required for EF-G turnover.fluorescence ͉ GTP-binding protein ͉ nucleotide exchange ͉ transient kinetics ͉ translation I n the translocation step of the elongation cycle, peptidyl-tRNA moves from the A site of the ribosome to the P site, carrying the mRNA along, and deacylated tRNA moves out of the P site into the E site from where it dissociates. Translocation is promoted by elongation factor G (EF-G), a large, five-domain GTPase that hydrolyzes GTP during the reaction. According to pre-steady-state kinetic analyses, EF-G binds to the pretranslocation ribosome in the GTP-bound form and subsequent rapid GTP hydrolysis precedes translocation (1, 2). Further analyses revealed that GTP hydrolysis drives a rearrangement of the ribosome, referred to as unlocking, that precedes and limits the rate of tRNA-mRNA movement (3). When GTP hydrolysis is avoided by using nonhydrolyzable GTP analogs, such as GDPNP or GDPCP (4, 5), EF-G still promotes translocation, albeit at a 50-fold lower rate (1), and the dissociation of EF-G from the ribosome is slowed down by about the same factor (6). The release of inorganic phosphate (P i ) from ribosome-bound EF-G⅐GDP⅐P i is not required to drive translocation, because ribosomes containing mutant protein L7͞12, which are slow in P i release and in EF-G turnover, remain rapid in single-round translocation (3, 7). Thus, the driving force of EF-G action in ribosome unlocking is a conformational change that is induced by GTP hydrolysis. Subsequent P i release induces another conformational change that is required for EF-G to dissociate from the ribosome (7). GTP hydrolysis reduces the free energy of activation of translocation by Ϸ2.5 kcal͞mol. The difference results from a large decrease of the activation enthalpy and a sma...

Elongation factors (EFs) Tu and G are GTPases that have important functions in protein synthesis. The low intrinsic GTPase activity of both factors is strongly stimulated on the ribosome by unknown mechanisms. Here we report that isolated ribosomal protein L7/12 strongly stimulates GTP hydrolysis by EF-G, but not by EF-Tu, indicating a major contribution of L7/12 to GTPase activation of EF-G on the ribosome. The effect is due to the acceleration of the catalytic step because the rate of GDP-GTP exchange on EF-G, as measured by rapid kinetics, is much faster than the steady-state GTPase rate. The unique, highly conserved arginine residue in the C-terminal domain of L7/12 is not essential for the activation, excluding an "arginine finger"-type mechanism. L7/12 appears to function by stabilizing the GTPase transition state of EF-G.

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