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The ribosome uses two active mechanisms to unwind messenger RNA during translation

Nature volume 475, pages 118–121 (2011)Cite this article

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Abstract

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting1,2, the modulation of protein expression levels3,4, ribosome localization5 and co-translational protein folding6. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases7, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily1,2. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.

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Figure 1: Experimental set-up.
Figure 2: Dependence of translation rate on force and mRNA GC content.
Figure 3: The molecular arrangement of a translocating ribosome.

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Acknowledgements

We thank members of the Tinoco and Bustamante labs for helpful discussions, especially S. B. Smith for his help with the optical tweezers, J. Moffitt for advice on data analysis and C. Kaiser for suggestions. Our work was supported by grants from the National Institutes of Health (to I.T., C.B. and H.F.N.) and the Human Frontiers Science Program (to I.T. and H.F.N.).

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Author notes

  1. Jin-Der Wen

    Present address: Present address: Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 10617, Taiwan.,

Authors and Affiliations

  1. Jason L. Choy Laboratory of Single Molecule Biophysics and QB3 Institute, University of California, Berkeley, 94720, California, USA

    Xiaohui Qu, Jin-Der Wen & Carlos Bustamante

  2. Department of Chemistry, University of California, Berkeley, 94720, California, USA

    Xiaohui Qu, Jin-Der Wen, Carlos Bustamante & Ignacio Tinoco

  3. Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, 95064, California, USA

    Laura Lancaster & Harry F. Noller

  4. Department of Molecular and Cellular Biology, Department of Physics and Howard Hughes Medical Institute, University of California, Berkeley, 94720, California, USA

    Carlos Bustamante

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Contributions

X.Q. and J.-D.W. conducted the experiments and performed the analysis; X.Q., J.-D.W. and L.L. prepared and provided experimental materials; and all authors helped to write the paper.

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Correspondence to Carlos Bustamante or Ignacio Tinoco.

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The authors declare no competing financial interests.

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The file contains Supplementary Figures 1-8 with legends, Supplementary Tables 1-2, a Supplementary Discussion and additional references. (PDF 614 kb)

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Qu, X., Wen, JD., Lancaster, L. et al. The ribosome uses two active mechanisms to unwind messenger RNA during translation. Nature 475, 118–121 (2011). https://doi.org/10.1038/nature10126

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