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Spontaneous reverse movement of mRNA-bound tRNA through the ribosome

Nature Structural & Molecular Biology volume 14, pages 318–324 (2007)Cite this article

Abstract

During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA–mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA–mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.

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Figure 1: Properties of the post-translocation complex.
Figure 2: Retrotranslocation of fMetVal-tRNAVal.
Figure 3: Requirements of retrotranslocation with respect to E-site tRNA.
Figure 4: Retrotranslocation of fMetPhe-tRNAPhe.
Figure 5: Three-dimensional cryo-EM reconstructions of tRNA-ribosome complexes before and after retrotranslocation.

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Acknowledgements

We thank T. Pestova (SUNY) and A. Kubarenko for help with the toeprinting analysis and C. Schillings, A. Böhm, S. Möbitz, P. Striebeck, W. Jahn and S. Kraffzig for expert technical assistance. Work in our laboratories is supported by the Deutsche Forschungsgemeinschaft (M.V.R. and W.W.), the Fonds der Chemischen Industrie (M.V.R. and W.W.), the Alfried Krupp von Bohlen und Halbach-Stiftung (M.V.R. and W.W.), the Federal Ministry of Education and Research (BMBF), Germany (H.S.), the Russian Foundation for Basic Research (Y.P.S.) and the International Bureau of the BMBF (W.W. and Y.P.S.). N.F. is supported by a Boehringer-Ingelheim fellowship.

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Author notes

  1. Andrey L Konevega and Niels Fischer: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Physical Biochemistry, University of Witten/Herdecke, Witten, 58448, Germany

    Andrey L Konevega & Marina V Rodnina

  2. 3D Electron Cryomicroscopy Group, Max-Planck-Institute for Biophysical Chemistry, Göttingen, 37077, Germany

    Niels Fischer & Holger Stark

  3. Petersburg Nuclear Physics Institute, Russian Academy of Sciences, Gatchina, 188300, Russia

    Yuri P Semenkov

  4. Institute of Molecular Biology, University of Witten/Herdecke, Witten, 58448, Germany

    Wolfgang Wintermeyer

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  1. Andrey L Konevega
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Contributions

M.V.R., Y.P.S., W.W. and H.S. designed experiments, A.L.K. and Y.P.S. performed biochemical experiments, N.F. performed the cryo-EM analysis, M.V.R. and W.W. wrote the manuscript draft and all authors contributed to the final version of the manuscript.

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Correspondence to Wolfgang Wintermeyer or Marina V Rodnina.

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Konevega, A., Fischer, N., Semenkov, Y. et al. Spontaneous reverse movement of mRNA-bound tRNA through the ribosome. Nat Struct Mol Biol 14, 318–324 (2007). https://doi.org/10.1038/nsmb1221

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