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Natural amino acids do not require their native tRNAs for efficient selection by the ribosome

Nature Chemical Biology volume 5, pages 947–953 (2009)Cite this article

Abstract

The involvement of tRNA structural elements beyond the anticodon in aminoacyl-tRNA (aa-tRNA) selection by the ribosome has revealed that substrate recognition is considerably more complex than originally envisioned in the adaptor hypothesis. By combining recent breakthroughs in aa-tRNA synthesis and mechanistic and structural studies of protein synthesis, we have investigated whether aa-tRNA recognition further extends to the amino acid, which would explain various translation disorders exhibited by misacylated tRNAs. Contrary to expectation, we find that natural amino acids misacylated onto natural but non-native tRNAs are selected with efficiencies very similar to those of their correctly acylated counterparts. Despite this, small but reproducible differences in selection indeed demonstrate that the translational machinery is sensitive to the amino acid–tRNA pairing. These results suggest either that the ribosome is an exquisite sensor of natural versus unnatural amino acid–tRNA pairings and/or that aa-tRNA selection is not the primary step governing the amino acid specificity of the ribosome.

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Figure 1: Design and synthesis of aminoacyl-tRNAs.
Figure 2: Dipeptide yield and competition translations.
Figure 3: aa-tRNA dynamics during selection of correctly acylated and misacylated tRNAs.

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References

  1. Ledoux, S. & Uhlenbeck, O.C. Different aa-tRNAs are selected uniformly on the ribosome. Mol. Cell 31, 114–123 (2008).

    Article  CAS  Google Scholar 

  2. Ogle, J.M. & Ramakrishnan, V. Structural insights into translational fidelity. Annu. Rev. Biochem. 74, 129–177 (2005).

    Article  CAS  Google Scholar 

  3. Rodnina, M.V. & Wintermeyer, W. Fidelity of aminoacyl-tRNA selection on the ribosome: kinetic and structural mechanisms. Annu. Rev. Biochem. 70, 415–435 (2001).

    Article  CAS  Google Scholar 

  4. Crick, F.H. On protein synthesis. Symp. Soc. Exp. Biol. 12, 138–163 (1958).

    CAS  PubMed  Google Scholar 

  5. Ramakrishnan, V. Ribosome structure and the mechanism of translation. Cell 108, 557–572 (2002).

    Article  CAS  Google Scholar 

  6. Chapeville, F. et al. On the role of soluble ribonucleic acid in coding for amino acids. Proc. Natl. Acad. Sci. USA 48, 1086–1092 (1962).

    Article  CAS  Google Scholar 

  7. Pape, T., Wintermeyer, W. & Rodnina, M.V. Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome. EMBO J. 17, 7490–7497 (1998).

    Article  CAS  Google Scholar 

  8. Kennell, D. & Riezman, H. Transcription and translation initiation frequencies of Escherichia-coli Lac operon. J. Mol. Biol. 114, 1–21 (1977).

    Article  CAS  Google Scholar 

  9. Marshall, R.A., Aitken, C.E., Dorywalska, M. & Puglisi, J.D. Translation at the single-molecule level. Annu. Rev. Biochem. 77, 177–203 (2008).

    Article  CAS  Google Scholar 

  10. Rodnina, M.V. & Wintermeyer, W. Ribosome fidelity: tRNA discrimination, proofreading and induced fit. Trends Biochem. Sci. 26, 124–130 (2001).

    Article  CAS  Google Scholar 

  11. Gromadski, K.B. & Rodnina, M.V. Kinetic determinants of high-fidelity tRNA discrimination on the ribosome. Mol. Cell 13, 191–200 (2004).

    Article  CAS  Google Scholar 

  12. Hirsh, D. & Gold, L. Translation of the UGA triplet in vitro by tryptophan transfer RNA's. J. Mol. Biol. 58, 459–468 (1971).

    Article  CAS  Google Scholar 

  13. Cochella, L. & Green, R. An active role for tRNA in decoding beyond codon:anticodon pairing. Science 308, 1178–1180 (2005).

    Article  CAS  Google Scholar 

  14. Ledoux, S., Olejniczak, M. & Uhlenbeck, O.C. A sequence element that tunes Escherichia coli tRNA(Ala)(GGC) to ensure accurate decoding. Nat. Struct. Mol. Biol. 16, 359–364 (2009).

    Article  CAS  Google Scholar 

  15. Murakami, H., Ohta, A. & Suga, H. Bases in the anticodon loop of tRNA(Ala)(GGC) prevent misreading. Nat. Struct. Mol. Biol. 16, 353–358 (2009).

    Article  CAS  Google Scholar 

  16. Blanchard, S.C., Gonzalez, R.L. Jr., Kim, H.D., Chu, S. & Puglisi, J.D. tRNA selection and kinetic proofreading in translation. Nat. Struct. Mol. Biol. 11, 1008–1014 (2004).

    Article  CAS  Google Scholar 

  17. Schuette, J.C. et al. GTPase activation of elongation factor EF-Tu by the ribosome during decoding. EMBO J. 28, 755–765 (2009).

    Article  CAS  Google Scholar 

  18. Villa, E. et al. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis. Proc. Natl. Acad. Sci. USA 106, 1063–1068 (2009).

    Article  CAS  Google Scholar 

  19. Piepenburg, O. et al. Intact aminoacyl-tRNA is required to trigger GTP hydrolysis by elongation factor Tu on the ribosome. Biochemistry 39, 1734–1738 (2000).

    Article  CAS  Google Scholar 

  20. Cornish, V.W. et al. Site-specific incorporation of biophysical probes into proteins. Proc. Natl. Acad. Sci. USA 91, 2910–2914 (1994).

    Article  CAS  Google Scholar 

  21. Ellman, J.A., Mendel, D. & Schultz, P.G. Site-specific incorporation of novel backbone structures into proteins. Science 255, 197–200 (1992).

    Article  CAS  Google Scholar 

  22. Cornish, V.W., Mendel, D. & Schultz, P.G. Probing protein structure and function with an expanded genetic code. Angew. Chem. Int. Edn Engl. 34, 621–633 (1995).

    Article  CAS  Google Scholar 

  23. Dougherty, D.A. Unnatural amino acids as probes of protein structure and function. Curr. Opin. Chem. Biol. 4, 645–652 (2000).

    Article  CAS  Google Scholar 

  24. Link, A.J., Mock, M.L. & Tirrell, D.A. Non-canonical amino acids in protein engineering. Curr. Opin. Biotechnol. 14, 603–609 (2003).

    Article  CAS  Google Scholar 

  25. Tan, Z., Forster, A.C., Blacklow, S.C. & Cornish, V.W. Amino acid backbone specificity of the Escherichia coli translation machinery. J. Am. Chem. Soc. 126, 12752–12753 (2004).

    Article  CAS  Google Scholar 

  26. Wang, L., Xie, J. & Schultz, P.G. Expanding the genetic code. Annu. Rev. Biophys. Biomol. Struct. 35, 225–249 (2006).

    Article  Google Scholar 

  27. Cload, S.T., Liu, D.R., Froland, W.A. & Schultz, P.G. Development of improved tRNAs for in vitro biosynthesis of proteins containing unnatural amino acids. Chem. Biol. 3, 1033–1038 (1996).

    Article  CAS  Google Scholar 

  28. LaRiviere, F.J., Wolfson, A.D. & Uhlenbeck, O.C. Uniform binding of aminoacyl-tRNAs to elongation factor Tu by thermodynamic compensation. Science 294, 165–168 (2001).

    Article  CAS  Google Scholar 

  29. Dale, T., Fahlman, R.P., Olejniczak, M. & Uhlenbeck, O.C. Specificity of the ribosomal A site for aminoacyl-tRNAs. Nucleic Acids Res. 37, 1202–1210 (2009).

    Article  CAS  Google Scholar 

  30. Lee, J.W. et al. Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration. Nature 443, 50–55 (2006).

    Article  CAS  Google Scholar 

  31. Murakami, H., Ohta, A., Ashigai, H. & Suga, H. A highly flexible tRNA acylation method for non-natural polypeptide synthesis. Nat. Methods 3, 357–359 (2006).

    Article  CAS  Google Scholar 

  32. Sprinzl, M., Hartmann, T., Weber, J., Blank, J. & Zeidler, R. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 17 (suppl.): r1–r172 (1989).

    Article  CAS  Google Scholar 

  33. Asahara, H. & Uhlenbeck, O.C. Predicting the binding affinities of misacylated tRNAs for Thermus thermophilus EF-Tu.GTP. Biochemistry 44, 11254–11261 (2005).

    Article  CAS  Google Scholar 

  34. Gonzalez, R.L. Jr., Chu, S. & Puglisi, J.D. Thiostrepton inhibition of tRNA delivery to the ribosome. RNA 13, 2091–2097 (2007).

    Article  CAS  Google Scholar 

  35. Lee, T.H., Blanchard, S.C., Kim, H.D., Puglisi, J.D. & Chu, S. The role of fluctuations in tRNA selection by the ribosome. Proc. Natl. Acad. Sci. USA 104, 13661–13665 (2007).

    Article  CAS  Google Scholar 

  36. Pavlov, M.Y. et al. Slow peptide bond formation by proline and other N-alkylamino acids in translation. Proc. Natl. Acad. Sci. USA 106, 50–54 (2009).

    Article  CAS  Google Scholar 

  37. Curnow, A.W. et al. Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation. Proc. Natl. Acad. Sci. USA 94, 11819–11826 (1997).

    Article  CAS  Google Scholar 

  38. Crick, F.H.C. The recent excitement in the coding problem. Prog. Nucleic Acid Res. Mol. Biol. 1, 163–217 (1963).

    Article  CAS  Google Scholar 

  39. Wohlgemuth, I., Brenner, S., Beringer, M. & Rodnina, M.V. Modulation of the rate of peptidyl transfer on the ribosome by the nature of substrates. J. Biol. Chem. 283, 32229–32235 (2008).

    Article  CAS  Google Scholar 

  40. Zaher, H.S. & Green, R. Quality control by the ribosome following peptide bond formation. Nature 457, 161–166 (2009).

    Article  CAS  Google Scholar 

  41. Beringer, M. Modulating the activity of the peptidyl transferase center of the ribosome. RNA 14, 795–801 (2008).

    Article  CAS  Google Scholar 

  42. Wang, J., Xie, J. & Schultz, P.G. A genetically encoded fluorescent amino acid. J. Am. Chem. Soc. 128, 8738–8739 (2006).

    Article  CAS  Google Scholar 

  43. Rothman, D.M. et al. Caged phosphoproteins. J. Am. Chem. Soc. 127, 846–847 (2005).

    Article  CAS  Google Scholar 

  44. Forster, A.C. et al. Programming peptidomimetic syntheses by translating genetic codes designed de novo. Proc. Natl. Acad. Sci. USA 100, 6353–6357 (2003).

    Article  CAS  Google Scholar 

  45. Ohta, A., Murakami, H., Higashimura, E. & Suga, H. Synthesis of polyester by means of genetic code reprogramming. Chem. Biol. 14, 1315–1322 (2007).

    Article  CAS  Google Scholar 

  46. Subtelny, A.O., Hartman, M.C. & Szostak, J.W. Ribosomal synthesis of N-methyl peptides. J. Am. Chem. Soc. 130, 6131–6136 (2008).

    Article  CAS  Google Scholar 

  47. Dedkova, L.M., Fahmi, N.E., Golovine, S.Y. & Hecht, S.M. Construction of modified ribosomes for incorporation of D-amino acids into proteins. Biochemistry 45, 15541–15551 (2006).

    Article  CAS  Google Scholar 

  48. Doi, Y., Ohtsuki, T., Shimizu, Y., Ueda, T. & Sisido, M. Elongation factor Tu mutants expand amino acid tolerance of protein biosynthesis system. J. Am. Chem. Soc. 129, 14458–14462 (2007).

    Article  CAS  Google Scholar 

  49. Spahn, C.M. & Prescott, C.D. Throwing a spanner in the works: antibiotics and the translation apparatus. J. Mol. Med. 74, 423–439 (1996).

    Article  CAS  Google Scholar 

  50. Blanchard, S.C., Kim, H.D., Gonzalez, R.L. Jr., Puglisi, J.D. & Chu, S. tRNA dynamics on the ribosome during translation. Proc. Natl. Acad. Sci. USA 101, 12893–12898 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by funds to V.W.C. and R.L.G. Jr. from Columbia University and by grants to R.L.G. Jr. from the Burroughs Wellcome Fund (CABS 1004856) and T.S.L. from the US National Institutes of Health (RO1 GM54469). P.R.E. was supported by Columbia University College of Physicians and Surgeons' MD/PhD Program (US National Institutes of Health Institutional Training Grant T32 GM07367). We thank Y.-M. Hou and M. Dupasquier (both at Thomas Jefferson University) for the kind gift of a nucleotidyl transferase overexpression strain, D. Tirrell (California Institute of Technology) for the gift of a phenylalanyl-tRNA synthetase overexpressing strain and N. Prywes (Columbia University) for cloning and purifying alanyl-tRNA synthetase as well as aiding in tRNAAla purification. We also thank H. Suga, R. Green, H. Zaher and L.G. Dickson for helpful discussions and advice. Finally, we are indebted to V. Mondol and S. Das for managing the Cornish and Gonzalez laboratories, respectively.

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Authors and Affiliations

  1. Integrated Program in Cellular, Molecular and Biomedical Sciences, Columbia University, New York, New York, USA

    Philip R Effraim & Michael T Englander

  2. Department of Chemistry, Columbia University, New York, New York, USA

    Philip R Effraim, Jiangning Wang, Michael T Englander, Josh Avins, Ruben L Gonzalez Jr & Virginia W Cornish

  3. Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA

    Thomas S Leyh

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  1. Philip R Effraim
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Contributions

P.R.E., T.S.L., R.L.G. and V.W.C. designed research; P.R.E. and J.W. performed research; M.T.E. and J.A. contributed reagents; P.R.E. and J.W. analyzed data; and P.R.E., T.S.L., R.L.G. and V.W.C. wrote the paper.

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Correspondence to Thomas S Leyh, Ruben L Gonzalez Jr or Virginia W Cornish.

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Effraim, P., Wang, J., Englander, M. et al. Natural amino acids do not require their native tRNAs for efficient selection by the ribosome. Nat Chem Biol 5, 947–953 (2009). https://doi.org/10.1038/nchembio.255

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