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Aminoglycoside activity observed on single pre-translocation ribosome complexes

Nature Chemical Biology volume 6, pages 54–62 (2010)Cite this article

An Erratum to this article was published on 01 March 2010

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Abstract

Aminoglycoside-class antibiotics bind directly to ribosomal RNA, imparting pleiotropic effects on ribosome function. Despite in-depth structural investigations of aminoglycoside–RNA oligonucleotide and aminoglycoside-ribosome interactions, mechanisms explaining the unique ribosome inhibition profiles of chemically similar aminoglycosides remain elusive. Here, using single-molecule fluorescence resonance energy transfer (smFRET) methods, we show that high-affinity aminoglycoside binding to the conserved decoding site region of the functional pre-translocation ribosome complex specifically remodels the nature of intrinsic dynamic processes within the particle. The extents of these effects, which are distinct for each member of the aminoglycoside class, strongly correlate with their inhibition of EF-G–catalyzed translocation. Neomycin, a 4,5-linked aminoglycoside, binds with lower affinity to one or more secondary binding sites, mediating distinct structural and dynamic perturbations that further enhance translocation inhibition. These new insights help explain why closely related aminoglycosides elicit pleiotropic translation activities and demonstrate the potential utility of smFRET as a tool for dissecting the mechanisms of antibiotic action.

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Figure 1: Aminoglycoside-induced restructuring of the ribosome 16S decoding site.
Figure 2: Kanamycin binding to the decoding site increases time-averaged occupancy of specific ribosome conformations.
Figure 3: Changes in population time-averaged state occupancy are caused by drug-induced changes in single ribosomes.
Figure 4: Alterations in tRNA dynamics are a common effect of aminoglycoside binding to the ribosome.
Figure 5: Neomycin alters tRNA dynamics on the ribosome in a bimodal fashion.
Figure 6: Stabilization of the classical state is strongly correlated with inhibition of translocation by decoding site–binding aminoglycosides.
Figure 7: Kinetic scheme of aminoglycoside-induced changes in tRNA dynamics.

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  • 12 February 2010

    In the version of this article initially published, in the key for Figure 6a, the units for two of the inhibitor concentrations were incorrectly labeled as “M”, where they should have read “µM”. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

The authors thank J. Munro for comments and suggestions during the preparation of this manuscript and for the generation of ribosome structural diagrams. We thank all members of the laboratory for their constructive comments during preparation of this manuscript, as well as R. Green (Johns Hopkins School of Medicine, Howard Hughes Medical Institute) for providing MS2-tagged rRNA constructs. This work was supported by the US National Institute of General Medical Sciences (5R01GM079238-03), New York State Foundation for Science, Technology and Innovation, and a US National Science Foundation CAREER award. M.B.F. is a trainee in the Weill Cornell/Rockefeller University/Sloan-Kettering Tri-Institutional MD-PhD Program supported by US National Institutes of Health Medical Scientist Training Program grant GM07739. D.S.T. is supported by the Tri-Institutional Training Program in Computational Biology and Medicine.

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  1. These authors contributed equally to this work.

Authors and Affiliations

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

    Michael B Feldman, Daniel S Terry, Roger B Altman & Scott C Blanchard

  2. Weill Cornell/Rockefeller University/Sloan-Kettering Tri-Institutional MD-PhD Program, New York, New York, USA

    Michael B Feldman

  3. Tri-Institutional Training Program in Computational Biology and Medicine, New York, New York, USA

    Daniel S Terry

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Contributions

M.B.F. and S.C.B. designed the research. M.B.F. performed the smFRET experiments. D.S.T. implemented the automated analysis software, and D.S.T. and M.B.F. performed the data analysis. R.B.A. prepared the dye-labeled tRNA and ribosome complexes and reagents for the smFRET experiments. M.B.F., D.S.T. and S.C.B. prepared the manuscript.

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Correspondence to Scott C Blanchard.

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Feldman, M., Terry, D., Altman, R. et al. Aminoglycoside activity observed on single pre-translocation ribosome complexes. Nat Chem Biol 6, 54–62 (2010). https://doi.org/10.1038/nchembio.274

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