Book of Abstracts: Albany 2007
June 19-23 2007
Structures of eEF2-80S Ribosome Complexes Reveal the Role of GTP Hydrolysis in Translocation
Eukaryotic elongation factor 2 (eEF2; EF-G in eubacteria) is a unique GTPase responsible for the translocation of the tRNA-mRNA duplex through the ribosome during the elongation cycle of protein synthesis. Cryo-EM reconstructions have revealed that eEF2 (1) and EF-G (2, 3) undergo gross conformational changes upon binding to the ribosome, which causes a ratchet-like rotation of the small ribosomal subunit with respect to the large ribosomal subunit. While these conformational changes apparently contribute to the forward movement of the tRNA-mRNA complex, pre-steady state kinetic studies argue that translocation occurs only after GTP hydrolysis (4, 5). To understand the effects of eEF2 binding and that of GTP hydrolysis in translocation, we have performed cryo-EM reconstructions of 80S·eEF2 complexes in states represented as immediately before (GDPNP) and immediately after (GDP:sordarin) GTP hydrolysis. We also used the ADP-ribosyl (ADPR) moiety of ADPR-eEF2 as a density marker to track structural changes in the conserved tip region of domain IV in eEF2 as a function of GTP hydrolysis. In the absence of A-site tRNA, eEF2 and ADPR-eEF2 bind to the ribosome similarly, with the main difference in the maps being that the ADPR-eEF2 reconstruction shows clear density that can be attributed to the ADPR modification. The study revealed that the critical Switch 1 loop of 80S-bound eEF2 is ordered in the GTP-bound state and becomes disordered upon GTP hydrolysis. Additionally, we observe subtle conformational changes in eEF2 upon GTP hydrolysis, specifically a 6 Å shift in the tip of domain IV, which is near the ribosomal decoding center. Interpretation of our structural data, combined with previously reported biochemical and kinetic data, suggests that GTP hydrolysis causes the tip of domain IV of eEF2 to sever the link between the ribosomal decoding center and the tRNA-mRNA duplex. We present a model of translocation describing how a head rotation of the small ribosomal subunit conducts the bulk of translocation and explain how this head rotation can only occur upon GTP hydrolysis of eEF2/EF-G. Thus, our study reconciles the results of the kinetic measurements with the previous cryo-EM data on eEF2/EF-G-ribosome interaction.
References and Footnotes
Derek J. Taylor1
1Howard Hughes Medical Institute