Article | Published:

mRNA translocation occurs during the second step of ribosomal intersubunit rotation

Nature Structural & Molecular Biology volume 18, pages 457462 (2011) | Download Citation

Abstract

During protein synthesis, mRNA and tRNA undergo coupled translocation through the ribosome in a process that is catalyzed by elongation factor G (EF-G). On the basis of cryo-EM reconstructions, counterclockwise and clockwise rotational movements between the large and small ribosomal subunits have been implicated in a proposed ratcheting mechanism to drive the unidirectional movement of translocation. We used a combination of two fluorescence-based approaches to study the timing of these events, intersubunit fluorescence resonance energy transfer measurements to observe relative rotational movement of the subunits, and a fluorescence quenching assay to monitor translocation of mRNA. Binding of EF-G–GTP first induces rapid counterclockwise intersubunit rotation, followed by a slower, clockwise reversal of the rotational movement. We compared the rates of these movements and found that mRNA translocation occurs during the second, clockwise rotation event, corresponding to the transition from the hybrid state to the classical state.

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Acknowledgements

These studies were supported by grant no. GM-17129 from the US National Institutes of Health, US National Science Foundation (NSF) grant no. MCB-0723300 (to H.F.N.), and a North Atlantic Treaty Organization–NSF postdoctoral fellowship to D.N.E. The authors thank D. Herschlag, M. Rodnina, S. Joseph, A. Korostelev and L. Lancaster for critical discussions.

Author information

Author notes

    • Dmitri N Ermolenko

    Present address: Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA.

Affiliations

  1. Center for Molecular Biology of RNA, University of California, Santa Cruz, Santa Cruz, California, USA.

    • Dmitri N Ermolenko
    •  & Harry F Noller
  2. Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, California, USA.

    • Dmitri N Ermolenko
    •  & Harry F Noller

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Contributions

D.N.E. and H.F.N. designed the research; D.N.E. carried out the experiments; D.N.E. and H.F.N. analyzed the data and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Harry F Noller.

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DOI

https://doi.org/10.1038/nsmb.2011

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