Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome

doi:10.1073/pnas.0606099103

Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome

Institutes of ^†Molecular Biology and
^‡Physical Biochemistry, University of Witten-Herdecke, D-58448 Witten, Germany

Communicated by Harry F. Noller, University of California, Santa Cruz, CA, July 19, 2006 (received for review March 24, 2006)

Abstract

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 GTP/GDP binding and show that EF-G binds GTP and GDP with affinities in the 20 to 40 μM 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-G·GDPNP 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 ribosome-complex 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 P_i release induces further rearrangements of both EF-G and the ribosome that are required for EF-G turnover.

Footnotes

^§To whom correspondence should be addressed. E-mail: winterme{at}uni-wh.de
Author contributions: B.W. and A.S. performed research; B.W., A.S., M.V.R., and W.W. analyzed data; M.V.R. and W.W. designed research; and M.V.R. and W.W. wrote the paper.
Conflict of interest statement: No conflicts declared.
Abbreviations:

EF-G,

elongation factor G;

mant,

(2′,3′)-O-(N-methylanthraniloyl).