Letter | Published:

Direct charging of tRNACUA with pyrrolysine in vitro and in vivo

Nature volume 431, pages 333335 (16 September 2004) | Download Citation

Subjects

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

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

  2. 2.

    , & Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science 296, 1459–1462 (2002)

  3. 3.

    & The 22nd amino acid. Science 296, 1409–1411 (2002)

  4. 4.

    , & 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)

  5. 5.

    , & Clustered genes encoding the methyltransferases of methanogenesis from monomethylamine. J. Bacteriol. 180, 3432–3440 (1998)

  6. 6.

    , , & 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)

  7. 7.

    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)

  8. 8.

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

  9. 9.

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

  10. 10.

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

  11. 11.

    , & 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)

  12. 12.

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

  13. 13.

    & 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)

  14. 14.

    , , & Aminoacyl-tRNA synthetases: versatile players in the changing theater of translation. RNA 8, 1363–1372 (2002)

  15. 15.

    & 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)

  16. 16.

    , , , & Substrate specificity of the periplasmic dipeptide-binding protein from Escherichia coli: experimental basis for the design of peptide prodrugs. Microbiology 145, 2891–2901 (1999)

  17. 17.

    , & Uniform binding of aminoacyl-tRNAs to elongation factor Tu by thermodynamic compensation. Science 294, 165–168 (2001)

  18. 18.

    Selenocysteine. Annu. Rev. Biochem. 65, 83–100 (1996)

  19. 19.

    & Selenocysteine inserting tRNAs: an overview. FEMS Microbiol. Rev. 23, 335–351 (1999)

  20. 20.

    , , & Expanding the genetic code of Escherichia coli. Science 292, 498–500 (2001)

  21. 21.

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

  22. 22.

    , & Identification of an expanded set of translationally active methionine analogues in Escherichia coli. FEBS Lett. 502, 25–30 (2001)

  23. 23.

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

  24. 24.

    & 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)

  25. 25.

    , , , & Nonorthologous replacement of lysyl-tRNA synthetase prevents addition of lysine analogues to the genetic code. Proc. Natl Acad. Sci. USA 100, 14351–14356 (2003)

  26. 26.

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

  27. 27.

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

Download references

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.

Author information

Author notes

    • Sherry K. Blight
    • , Ross C. Larue
    •  & Anirban Mahapatra

    These authors contributed equally to this work.

    • Gang Zhao

    Present address: Nitto Denko Technical Corporation, 401 Jones Road, Oceanside, California 92054, USA.

Affiliations

  1. Department of Microbiology, 484 West 12th Avenue, The Ohio State University, Columbus, Ohio 43210, USA

    • Sherry K. Blight
    • , Ross C. Larue
    • , Anirban Mahapatra
    • , David G. Longstaff
    • , Edward Chang
    •  & Joseph A. Krzycki
  2. Department of Chemistry, 100 West 18th Avenue, The Ohio State University, Columbus, Ohio 43210, USA

    • Gang Zhao
    •  & Michael K. Chan
  3. Department of Biochemistry, 484 West 12th Avenue, The Ohio State University, Columbus, Ohio 43210, USA

    • Michael K. Chan
  4. Ohio State University Biochemistry Program, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA

    • Patrick T. Kang
    • , Michael K. Chan
    •  & Joseph A. Krzycki
  5. CCIC/Mass Spectrometry and Proteomics Facility, The Ohio State University, 116 W 19th Ave, Columbus, Ohio 43210, USA

    • Kari B. Green-Church

Authors

  1. Search for Sherry K. Blight in:

  2. Search for Ross C. Larue in:

  3. Search for Anirban Mahapatra in:

  4. Search for David G. Longstaff in:

  5. Search for Edward Chang in:

  6. Search for Gang Zhao in:

  7. Search for Patrick T. Kang in:

  8. Search for Kari B. Green-Church in:

  9. Search for Michael K. Chan in:

  10. Search for Joseph A. Krzycki in:

Competing interests

The authors declare that they have no competing financial interests.

Corresponding author

Correspondence to Joseph A. Krzycki.

Supplementary information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature02895

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.