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Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation

Nature Structural & Molecular Biology volume 23, pages 333341 (2016) | Download Citation

Abstract

Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre–steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G–GDP–bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.

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Acknowledgements

We thank R. Green (Johns Hopkins University) for providing the S13-knockout strain and P. Schultz (Scripps Research Institute) for providing the L5-knockout strain. We also thank D. Terry of the Blanchard laboratory for assistance with three-color FRET data acquisition and analysis. This work was supported by the US National Institutes of Health (2R01GM079238 and 5R01GM098859 to S.C.B.).

Author information

Author notes

    • Michael R Wasserman
    •  & Jose L Alejo

    These authors contributed equally to this work.

Affiliations

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

    • Michael R Wasserman
    • , Jose L Alejo
    • , Roger B Altman
    •  & Scott C Blanchard
  2. Tri-Institutional Training Program in Chemical Biology, Weill Cornell Medical College, New York, New York, USA.

    • Scott C Blanchard

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Contributions

M.R.W. prepared dye-labeled ribosomes and EF-G. R.B.A. and M.R.W. prepared dye-labeled tRNAs. J.L.A. and M.R.W. performed the smFRET imaging. J.L.A. analyzed the smFRET results. J.L.A. and M.R.W. made the figures. S.C.B., M.R.W. and J.L.A. designed the study. All authors discussed the results and contributed to the writing of the manuscript.

Competing interests

S.C.B. and R.B.A. have an equity interest in Lumidyne Technologies.

Corresponding author

Correspondence to Scott C Blanchard.

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DOI

https://doi.org/10.1038/nsmb.3177

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