Abstract
Termination of protein synthesis by the ribosome requires two release factor (RF) classes. The class II RF3 is a GTPase that removes class I RFs (RF1 or RF2) from the ribosome after release of the nascent polypeptide1,2,3. RF3 in the GDP state binds to the ribosomal class I RF complex, followed by an exchange of GDP for GTP and release of the class I RF. As GTP hydrolysis triggers release of RF3 (ref. 4), we trapped RF3 on Escherichia coli ribosomes using a nonhydrolysable GTP analogue. Here we show by cryo-electron microscopy that the complex can adopt two different conformational states. In ‘state 1’, RF3 is pre-bound to the ribosome, whereas in ‘state 2’ RF3 contacts the ribosome GTPase centre. The transfer RNA molecule translocates from the peptidyl site in state 1 to the exit site in state 2. This translocation is associated with a large conformational rearrangement of the ribosome. Because state 1 seems able to accommodate simultaneously both RF3 and RF2, whose position is known from previous studies5,6, we can infer the release mechanism of class I RFs.
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Acknowledgements
We thank M. Ehrenberg and A. Zavialov for purified RF3 and 70S ribosome RCs; T. Pape for assistance in the early phase of this work; S. Chen, M. Schatz, R. Schmidt, and G. Willoughby for support; and R. Buckingham for sharing data before publication. B.P.K. thanks D. Moras, P. Schultz, M. Yusupov, G. Yusupova and B. Rees for support, discussions and comments on the manuscript; O. Poch for discussions on multiple sequence alignments; E. Orlova for initial image processing suggestions; and H. Saibil for the loan of a Tecnai 12 cryo-holder. The project was financed in part by grants from the Biotechnology and Biological Sciences Research Council, the European Union, and the Centre National de la Recherche Scientifique.
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M.v.H. is a shareholder of Image Science Software GmbH, distributors of the IMAGIC-5 software.
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Supplementary Figure 1
Principle of local multivariate statistical analysis exemplified on a typical view (side-view of the 70S ribosome); this approach was applied to all class averages (and to the particles included in these), and allowed particle separation enabling the analysis of two specific intermediate states. (JPG 39 kb)
Supplementary Figure 2
Some representative class averages (a and c) and their corresponding re-projections from the final 3D reconstructions (b and d) of states 1 and 2, respectively. (JPG 70 kb)
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Klaholz, B., Myasnikov, A. & van Heel, M. Visualization of release factor 3 on the ribosome during termination of protein synthesis. Nature 427, 862–865 (2004). https://doi.org/10.1038/nature02332
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DOI: https://doi.org/10.1038/nature02332
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