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Dynamics of translation by single ribosomes through mRNA secondary structures

Nature Structural & Molecular Biology volume 20, pages 582–588 (2013)Cite this article

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Abstract

During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we used single-molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem-loop or pseudoknot mRNA secondary structures. Downstream stem-loops containing 100% GC base pairs decrease the rates of both tRNA translocation within the ribosome and deacylated tRNA dissociation from the ribosomal exit site (E site). Downstream stem-loops or pseudoknots containing both GC and AU pairs also decrease the rate of tRNA dissociation, but they have little effect on tRNA translocation rate. Thus, somewhat unexpectedly, unfolding of mRNA secondary structures is more closely coupled to E-site tRNA dissociation than to tRNA translocation.

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Figure 1: mRNAs used in this study and their secondary structures.
Figure 2: Schematics of FRET studies on complexes formed through two elongation cycles that add arginine and phenylalanine to the nascent peptide at positions 8 and 9, respectively.
Figure 3: tRNA-tRNA smFRET.
Figure 4: L11-tRNA smFRET.
Figure 5: L1-tRNA smFRET.

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Acknowledgements

This work was supported by US National Institutes of Health grant R01GM080376 to B.S.C. and Y.E.G. and American Heart Association Postdoctoral Fellowship 12POST8910014 to C.C.

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Authors and Affiliations

  1. Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Chunlai Chen & Yale E Goldman

  2. Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Haibo Zhang, Michael Reiche, Ian Farrell & Barry S Cooperman

  3. Department of Biology, West Chester University of Pennsylvania, West Chester, Pennsylvania, USA

    Steven L Broitman

  4. Anima Cell Metrology, Inc., Bernardsville, New Jersey, USA

    Ian Farrell

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  1. Chunlai Chen
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Contributions

C.C., B.S.C. and Y.E.G. designed the experiments. C.C., S.L.B. and M.R. conducted the experiments and analyzed the data. H.Z. and I.F. prepared reagents. C.C., B.S.C. and Y.E.G. wrote the paper.

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Correspondence to Barry S Cooperman or Yale E Goldman.

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Chen, C., Zhang, H., Broitman, S. et al. Dynamics of translation by single ribosomes through mRNA secondary structures. Nat Struct Mol Biol 20, 582–588 (2013). https://doi.org/10.1038/nsmb.2544

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