| 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. It has been commonly understood that EF-G ˙ GTP enters the ribosome, GTP is hydrolyzed to GDP and following the release of EF-G ˙ GDP from the ribosome, GDP is spontaneously exchanged for GTP. New measurements of EF-G affinity for GTP/GDP have lead to the controversial proposal that EF-G ˙ GDP enters the ribosome whereupon GDP is exchanged for GTP [Zavialov, A.V., et al. J Biol, 2005. 4(2): p. 9.]. This assigns the ribosome a new functional role as a guanine exchange factor for EF-G. Additionally, it is proposed that EF-G in the GTP-bound form promotes movement of the tRNA-mRNA complex to an intermediate translocation state whereupon GTP is hydrolyzed, yielding the post-translocation state. Zavialov et al. report that this final step does not occur when GTP is substituted with a non-hydrolyzable GTP analog or GDP and contrasts to the model proposed by Wilden et al., where they report translocation at a reduced rate with non-hydrolyzable GTP analogs or GDP [Wilden, B., et al. Proc Natl Acad Sci USA, 2006. 103(37): p. 13670-5.].;Because Zavialov et al.'s reported measure of EF-G affinity to GTP contradicts those from other well-established groups, it is important to verify their findings. Additionally, the mechanistic model they propose for translocation lacks support from real-time kinetic data. In the present study, EF-G binding to guanine nucleotides has been characterized using both equilibrium and kinetic methods. Additionally, translocation has been measured in the presence of GTP and/or purified GDP, and by replacing GTP with a non-hydrolyzable analog. Our results indicate that EF-G binds GDP with 10-fold greater affinity over GTP, in accordance with earlier reports. Furthermore, GDP exchange on free EF-G is comparable to the rate observed in the presence of the ribosome and suggests the ribosome is not essential for nucleotide exchange. Additionally, we observe multiple changes in the translocation complex that occur upon EF-G ˙ GTP binding to the ribosome that are independent of GTP hydrolysis. |
