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Direct charging of tRNACUA with pyrrolysine in vitro and in vivo

Abstract

Pyrrolysine is the 22nd amino acid1,2,3. An unresolved question has been how this atypical genetically encoded residue is inserted into proteins, because all previously described naturally occurring aminoacyl-tRNA synthetases are specific for one of the 20 universally distributed amino acids. Here we establish that synthetic l-pyrrolysine is attached as a free molecule to tRNACUA by PylS, an archaeal class II aminoacyl-tRNA synthetase. PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNACUA in vitro in reactions specific for pyrrolysine. The addition of pyrrolysine to Escherichia coli cells expressing pylT (encoding tRNACUA) and pylS results in the translation of UAG in vivo as a sense codon. This is the first example from nature of direct aminoacylation of a tRNA with a non-canonical amino acid and shows that the genetic code of E. coli can be expanded to include UAG-directed pyrrolysine incorporation into proteins.

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Figure 1: Aminoacylation of tRNACUA in cellular tRNA pools monitored by acid-urea gel electrophoresis and northern blotting to detect tRNACUA.
Figure 2: Dependence on pyrrolysine of the 32PPi–ATP exchange reaction mediated by PylS-His6.
Figure 3: Anti-MtmB immunoblot of cell extracts of E. coli strains testing the suppression of the UAG codon in mtmB1.

References

  1. Hao, B. et al. A new UAG-encoded residue in the structure of a methanogen methyltransferase. Science 296, 1462–1466 (2002)

    ADS  CAS  Article  PubMed  Google Scholar 

  2. Srinivasan, G., James, C. M. & Krzycki, J. A. Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science 296, 1459–1462 (2002)

    ADS  CAS  Article  PubMed  Google Scholar 

  3. Atkins, J. F. & Gesteland, R. The 22nd amino acid. Science 296, 1409–1411 (2002)

    CAS  Article  PubMed  Google Scholar 

  4. Paul, L., Ferguson, D. J. & Krzycki, J. A. The trimethylamine methyltransferase gene and multiple dimethylamine methyltransferase genes of Methanosarcina barkeri contain in-frame and read-through amber codons. J. Bacteriol. 182, 2520–2529 (2000)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Burke, S. A., Lo, S. L. & Krzycki, J. A. Clustered genes encoding the methyltransferases of methanogenesis from monomethylamine. J. Bacteriol. 180, 3432–3440 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  6. James, C. M., Ferguson, T. K., Leykam, J. F. & Krzycki, J. A. The amber codon in the gene encoding the monomethylamine methyltransferase isolated from Methanosarcina barkeri is translated as a sense codon. J. Biol. Chem. 276, 34252–34258 (2001)

    CAS  Article  PubMed  Google Scholar 

  7. Polycarpo, C. et al. Activation of the pyrrolysine suppressor tRNA requires formation of a ternary complex with class I and class II lysyl-tRNA synthetases. Mol. Cell 12, 287–294 (2003)

    CAS  Article  PubMed  Google Scholar 

  8. Ibba, M. & Söll, D. Aminoacyl-tRNAs: setting the limits of the genetic code. Genes Dev. 18, 731–738 (2004)

    CAS  Article  PubMed  Google Scholar 

  9. Hao, B. et al. Reactivity and chemical synthesis of L-pyrrolysine – the 22nd amino acid. Chem. Biol. (in the press)

  10. Ho, Y. S. & Kan, Y. W. In vivo aminoacylation of human and Xenopus suppressor tRNAs constructed by site-specific mutagenesis. Proc. Natl Acad. Sci. USA 84, 2185–2188 (1987)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Varshney, U., Lee, C. P. & RajBhandary, U. L. Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase. J. Biol. Chem. 266, 24712–24718 (1991)

    CAS  PubMed  Google Scholar 

  12. Schimmel, P. & Söll, D. Aminoacyl-tRNA synthetases: general features and recognition of transfer RNAs. Annu. Rev. Biochem. 48, 601–648 (1979)

    CAS  Article  PubMed  Google Scholar 

  13. Cole, F. & Schimmel, P. R. On the rate law and mechanism of the adenosine triphosphate-pyrophosphate isotope exchange reaction of aminoacyl-transfer ribonucleic acid synthetases. Biochemistry 9, 480–489 (1970)

    CAS  Article  PubMed  Google Scholar 

  14. Francklyn, C., Perona, J. J., Puetz, J. & Hou, Y. M. Aminoacyl-tRNA synthetases: versatile players in the changing theater of translation. RNA 8, 1363–1372 (2002)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Burke, S. A. & Krzycki, J. A. Reconstitution of monomethylamine:coenzyme M methyl transfer with a corrinoid protein and two methyltransferases purified from Methanosarcina barkeri. J. Biol. Chem. 272, 16570–16577 (1997)

    CAS  Article  PubMed  Google Scholar 

  16. Smith, M. W., Tyreman, D. R., Payne, G. M., Marshall, N. J. & Payne, J. W. Substrate specificity of the periplasmic dipeptide-binding protein from Escherichia coli: experimental basis for the design of peptide prodrugs. Microbiology 145, 2891–2901 (1999)

    CAS  Article  PubMed  Google Scholar 

  17. 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)

    ADS  CAS  Article  PubMed  Google Scholar 

  18. Stadtman, T. C. Selenocysteine. Annu. Rev. Biochem. 65, 83–100 (1996)

    CAS  Article  PubMed  Google Scholar 

  19. Commans, S. & Bock, A. Selenocysteine inserting tRNAs: an overview. FEMS Microbiol. Rev. 23, 335–351 (1999)

    CAS  Article  PubMed  Google Scholar 

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

    ADS  CAS  Article  PubMed  Google Scholar 

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

    ADS  CAS  Article  PubMed  Google Scholar 

  22. Kiick, K. L., Weberskirch, R. & Tirrell, D. A. Identification of an expanded set of translationally active methionine analogues in Escherichia coli. FEBS Lett. 502, 25–30 (2001)

    CAS  Article  PubMed  Google Scholar 

  23. Chin, J. W. et al. An expanded eukaryotic genetic code. Science 301, 964–967 (2003)

    ADS  CAS  Article  PubMed  Google Scholar 

  24. Paul, L. & Krzycki, J. A. Sequence and transcript analysis of a novel Methanosarcina barkeri methyltransferase II homolog and its associated corrinoid protein homologous to methionine synthase. J. Bacteriol. 178, 6599–6607 (1996)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Jester, B. C., Levengood, J. D., Roy, H., Ibba, M. & Devine, K. M. Nonorthologous replacement of lysyl-tRNA synthetase prevents addition of lysine analogues to the genetic code. Proc. Natl Acad. Sci. USA 100, 14351–14356 (2003)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Sowers, K. R. & Schreier, H. J. in Archaea, a Laboratory Manual (eds Sowers, K. R. & Schreier, H. J.) 459–489 (Cold Spring Harbor Laboratory Press, Plainview, New York, 1995)

    Google Scholar 

  27. Wilm, M. et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466–469 (1996)

    ADS  CAS  Article  PubMed  Google Scholar 

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Acknowledgements

We thank J. Reeve, C. Daniels and J. Soares for discussions, R. L. Pitsch and N. M. Kleinholz at the Ohio State University's Campus Chemical Instrumentation Center for mass spectroscopic analyses, G. Srinivasan for construction of the lysS expression plasmid, and S. B. Smith for mass culture of methanogens.

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Correspondence to Joseph A. Krzycki.

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Blight, S., Larue, R., Mahapatra, A. et al. Direct charging of tRNACUA with pyrrolysine in vitro and in vivo. Nature 431, 333–335 (2004). https://doi.org/10.1038/nature02895

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