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A tRNA aminoacylation system for non-natural amino acids based on a programmable ribozyme

Nature Biotechnology volume 20, pages 723–728 (2002)Cite this article

Abstract

The ability to recognize tRNA identities is essential to the function of the genetic coding system. In translation aminoacyl-tRNA synthetases (ARSs) recognize the identities of tRNAs and charge them with their cognate amino acids. We show that an in vitro–evolved ribozyme can also discriminate between specific tRNAs, and can transfer amino acids to the 3′ ends of cognate tRNAs. The ribozyme interacts with both the CCA-3′ terminus and the anticodon loop of tRNAfMet, and its tRNA specificity is controlled by these interactions. This feature allows us to program the selectivity of the ribozyme toward specific tRNAs, and therefore to tailor effective aminoacyl-transfer catalysts. This method potentially provides a means of generating aminoacyl tRNAs that are charged with non-natural amino acids, which could be incorporated into proteins through cell-free translation.

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Figure 1: Strategy for the isolation of an acyl-transferase ribozyme that recognizes a specific tRNA.
Figure 2: Properties of the BC28 ribozyme.
Figure 3: Specificity of BC28 toward various tRNA substrates.
Figure 4: Programming tRNA specificity into the ribozyme.

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References

  1. Heckler, T.G. et al. T4 RNA ligase mediated preparation of novel “chemically misacylated” tRNAPhes. Biochemistry 23, 1468–1473 (1984).

    Article  CAS  Google Scholar 

  2. Robertson, S.A., Ellman, J.A. & Schultz, P.G. A general and efficient route for chemical aminoacylation of transfer RNAs. J.Am. Chem. Soc. 113, 2711–2729 (1991).

    Article  Google Scholar 

  3. Spirin, A.S. et al. Cell-free translation system capable of producing polypeptides in high yield. Science 242, 1162–1164 (1988).

    Article  CAS  Google Scholar 

  4. Kigawa, T. et al. Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett. 442, 15–19 (1999).

    Article  CAS  Google Scholar 

  5. Liu, D.R., Magliery, T.J., Pastrnak, M. & Schultz, P.G. Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo. Proc. Natl. Acad. Sci. USA 94, 10092–10097 (1997).

    Article  CAS  Google Scholar 

  6. Wang, L., Brock, A., Herberich, B. & Schultz, P.G. Expanding the genetic code of Escherichia coli. Science 292, 498–500 (2001).

    Article  CAS  Google Scholar 

  7. Döring, V. et al. Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway. Science 292, 501–504 (2001).

    Article  Google Scholar 

  8. Schimmel, P. & Söll, D. When protein engineering confronts the tRNA world. Proc. Natl. Acad. Sci. USA 94, 10007–10009 (1997).

    Article  CAS  Google Scholar 

  9. Liu, D.R. & Schultz, P.G. Progress toward the evolution of an organism with an expanded genetic code. Proc. Natl. Acad. Sci. USA 96, 4780–4785 (1999).

    Article  CAS  Google Scholar 

  10. Lohse, P.A. & Szostak, J.W. Ribozyme-catalyzed amino-acid transfer reactions. Nature 381, 442–444 (1996).

    Article  CAS  Google Scholar 

  11. Flynn-Charlebois, A., Lee, N. & Suga, H. A single metal ion plays structural and chemical roles in an acyl-transferase ribozyme. Biochemistry 40, 13623–13632 (2001).

    Article  CAS  Google Scholar 

  12. Lee, N., Bessho, Y., Wei, K., Szostak, J.W. & Suga, H. Ribozyme-catalyzed tRNA aminoacylation. Nat. Struct. Biol. 7, 28–33 (2000).

    Article  CAS  Google Scholar 

  13. Pütz, J. et al. Rapid selection of aminoacyl-tRNAs based on biotinylation of α-NH2 group of charged amino acids. Nucleic Acids Res. 25, 1862–1863 (1997).

    Article  Google Scholar 

  14. Mathews, D.H., Sabina, J., Zuker, M. & Turner, D.H. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911–940 (1999).

    Article  CAS  Google Scholar 

  15. Arslan, T., Mamaev, S.V., Mamaev, N.V. & Hecht, S.M. Structurally modified firefly luciferase. Effects of amino acid substitution at position 286. J. Am. Chem. Soc. 119, 10877–10887 (1997).

    Article  CAS  Google Scholar 

  16. Hohsaka, T., Ashizuka, Y., Murakami, H. & Sisido, M. Incorporation of nonnatural amino acids into streptavidin through in vitro frame-shift suppression. J. Am. Chem. Soc. 118, 9778–9779 (1996).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank all members of the Suga laboratory for helpful discussions, particularly N. Lee for synthesis of substrates. D.R.W.H. is a grateful recipient of a Royal Society–Fulbright Postdoctoral Fellowship (United Kingdom). This work was supported by US National Institutes of Health and National Science Foundation awards to H.S.

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  1. Department of Chemistry, University at Buffalo, State University of New York, Buffalo, 14260-3000, NY

    Yoshitaka Bessho, David R.W. Hodgson & Hiroaki Suga

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  1. Yoshitaka Bessho
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  3. Hiroaki Suga
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Correspondence to Hiroaki Suga.

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Bessho, Y., Hodgson, D. & Suga, H. A tRNA aminoacylation system for non-natural amino acids based on a programmable ribozyme. Nat Biotechnol 20, 723–728 (2002). https://doi.org/10.1038/nbt0702-723

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