Abstract
The ribosome converts genetic information into protein by selecting aminoacyl tRNAs whose anticodons base-pair to an mRNA codon. Mutations in the tRNA body can perturb this process and affect fidelity. The Hirsh suppressor is a well-studied tRNATrp harboring a G24A mutation that allows readthrough of UGA stop codons. Here we present crystal structures of the 70S ribosome complexed with EF-Tu and aminoacyl tRNA (native tRNATrp, G24A tRNATrp or the miscoding A9C tRNATrp) bound to cognate UGG or near-cognate UGA codons, determined at 3.2-Å resolution. The A9C and G24A mutations lead to miscoding by facilitating the distortion of tRNA required for decoding. A9C accomplishes this by increasing tRNA flexibility, whereas G24A allows the formation of an additional hydrogen bond that stabilizes the distortion. Our results also suggest that each native tRNA will adopt a unique conformation when delivered to the ribosome that allows accurate decoding.
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Acknowledgements
We thank R. Green and R. Ortiz-Meoz, Johns Hopkins University, for plasmids and bacterial strains for production of mutant tRNATrp, A. McCarthy and S. Brockhauser at ESRF ID14.4 for facilitating data collection, O. Uhlenbeck for helpful discussion and F. Murphy for scripting. This work was supported by the Medical Research Council, the Wellcome Trust, the Agouron Institute and the Louis-Jeantet Foundation. R.M.V. received support from the Gates-Cambridge scholarship. T.M.S. received support from the Human Frontier Science Program and Emmanuel College.
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T.M.S. and R.M.V. designed the experiments, prepared and crystallized the ribosomal complexes, collected and processed X-ray crystallography data, determined, refined and analyzed the structures, and prepared the manuscript and figures. A.C.K. purified macromolecular components, prepared and crystallized ribosomal complexes and aided with collection of X-ray crystallography data. V.R. suggested the study and provided intellectual input and supervision.
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V.R. is on the Senior Advisory Board of Rib-X Pharmaceuticals and both T.M.S. and V.R. hold stock options.
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Schmeing, T., Voorhees, R., Kelley, A. et al. How mutations in tRNA distant from the anticodon affect the fidelity of decoding. Nat Struct Mol Biol 18, 432–436 (2011). https://doi.org/10.1038/nsmb.2003
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DOI: https://doi.org/10.1038/nsmb.2003
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