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Coordinated conformational and compositional dynamics drive ribosome translocation

Nature Structural & Molecular Biology volume 20, pages 718–727 (2013)Cite this article

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Abstract

During translation elongation, the ribosome compositional factors elongation factor G (EF-G; encoded by fusA) and tRNA alternately bind to the ribosome to direct protein synthesis and regulate the conformation of the ribosome. Here, we use single-molecule fluorescence with zero-mode waveguides to directly correlate ribosome conformation and composition during multiple rounds of elongation at high factor concentrations in Escherichia coli. Our results show that EF-G bound to GTP (EF-G–GTP) continuously samples both rotational states of the ribosome, binding with higher affinity to the rotated state. Upon successful accommodation into the rotated ribosome, the EF-G–ribosome complex evolves through several rate-limiting conformational changes and the hydrolysis of GTP, which results in a transition back to the nonrotated state and in turn drives translocation and facilitates release of both EF-G–GDP and E-site tRNA. These experiments highlight the power of tracking single-molecule conformation and composition simultaneously in real time.

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Figure 1: Correlating conformation and composition with nonfluorescent FRET acceptor and ZMW.
Figure 2: Ribosome conformation drives translocation and regulates tRNA dynamics.
Figure 3: EF-G regulates ribosome conformational dynamics.
Figure 4: Role of EF-G–GTP hydrolysis.
Figure 5: Conformational selection for EF-G.
Figure 6: State-specific dynamics of EF-G to different ribosome conformations.
Figure 7: Perturbations of EF-G dynamics by antibiotics.
Figure 8: Ribosomal elongation.

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Acknowledgements

We would like to thank J. Korlach (Pacific Biosciences) for providing technical support on the ZMW instrumentation. This work was supported by US National Institutes of Health grants GM51266 (J.C., A.T. and J.D.P.) and GM099687 (A.P., S.E.O. and J.D.P.) and by a Stanford Interdisciplinary Graduate Fellowship (J.C.).

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Authors and Affiliations

  1. Department of Applied Physics, Stanford University, Stanford, California, USA

    Jin Chen & Albert Tsai

  2. Department of Structural Biology, Stanford University School of Medicine, Stanford, California, USA

    Jin Chen, Alexey Petrov, Albert Tsai, Seán E O'Leary & Joseph D Puglisi

  3. Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Stanford, California, USA

    Joseph D Puglisi

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  1. Jin Chen
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Contributions

J.C. performed all experiments and data analysis. J.C. and J.D.P. designed the project and wrote the manuscript. J.C., A.P., A.T., S.E.O. and J.D.P. discussed results. S.E.O. assisted with protein purification and labeling.

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Correspondence to Joseph D Puglisi.

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Chen, J., Petrov, A., Tsai, A. et al. Coordinated conformational and compositional dynamics drive ribosome translocation. Nat Struct Mol Biol 20, 718–727 (2013). https://doi.org/10.1038/nsmb.2567

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