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Correlated conformational events in EF-G and the ribosome regulate translocation

Nature Structural & Molecular Biology volume 17, pages 1470–1477 (2010)Cite this article

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Abstract

In bacteria, the translocation of tRNA and mRNA with respect to the ribosome is catalyzed by the conserved GTPase elongation factor-G (EF-G). To probe the rate-determining features in this process, we imaged EF-G–catalyzed translocation from two unique structural perspectives using single-molecule fluorescence resonance energy transfer. The data reveal that the rate at which the ribosome spontaneously achieves a transient, 'unlocked' state is closely correlated with the rate at which the tRNA-like domain IV-V element of EF-G engages the A site. After these structural transitions, translocation occurs comparatively fast, suggesting that conformational processes intrinsic to the ribosome determine the rate of translocation. Experiments conducted in the presence of non-hydrolyzable GTP analogs and specific antibiotics further reveal that allosterically linked conformational events in EF-G and the ribosome mediate rapid, directional substrate movement and EF-G release.

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Figure 1: Structural models of the ribosome and EF-G.
Figure 2: Observation of the translocation reaction from two unique structural perspectives.
Figure 3: The kinetics of unlocked-state formation and decay and EF-G–ribosome interactions are correlated.
Figure 4: A step-like increase in Cy3 fluorescence accompanies peptidyl-tRNA movement to the P site.
Figure 5: Translocation in the presence of inhibitors.
Figure 6

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Acknowledgements

The authors wish to thank C. Walsh (Harvard University) and members of his laboratory for providing the Sfp expression vector and for their assistance with the fluorescent labeling of EF-G and K.Y. Sanbonmatsu and P. Whitford for structural models of the E. coli ribosome. This work was supported by US National Institutes of Health grant GM079238.

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  1. Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, New York, USA

    James B Munro, Michael R Wasserman, Roger B Altman, Leyi Wang & Scott C Blanchard

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Contributions

J.B.M. and S.C.B. designed the experiments. J.B.M. and M.R.W. conducted the experiments and analyzed the data. R.B.A. and L.W. prepared reagents. J.B.M., M.R.W., R.B.A. and S.C.B. wrote the paper.

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Correspondence to Scott C Blanchard.

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Munro, J., Wasserman, M., Altman, R. et al. Correlated conformational events in EF-G and the ribosome regulate translocation. Nat Struct Mol Biol 17, 1470–1477 (2010). https://doi.org/10.1038/nsmb.1925

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