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Direct charging of tRNACUA with pyrrolysine in vitro and in vivo

Author

Listed:
  • Sherry K. Blight

    (The Ohio State University)

  • Ross C. Larue

    (The Ohio State University)

  • Anirban Mahapatra

    (The Ohio State University)

  • David G. Longstaff

    (The Ohio State University)

  • Edward Chang

    (The Ohio State University)

  • Gang Zhao

    (The Ohio State University
    Nitto Denko Technical Corporation)

  • Patrick T. Kang

    (Ohio State University Biochemistry Program, The Ohio State University)

  • Kari B. Green-Church

    (The Ohio State University)

  • Michael K. Chan

    (The Ohio State University
    The Ohio State University
    Ohio State University Biochemistry Program, The Ohio State University)

  • Joseph A. Krzycki

    (The Ohio State University
    Ohio State University Biochemistry Program, The Ohio State University)

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.

Suggested Citation

  • Sherry K. Blight & Ross C. Larue & Anirban Mahapatra & David G. Longstaff & Edward Chang & Gang Zhao & Patrick T. Kang & Kari B. Green-Church & Michael K. Chan & Joseph A. Krzycki, 2004. "Direct charging of tRNACUA with pyrrolysine in vitro and in vivo," Nature, Nature, vol. 431(7006), pages 333-335, September.
    • Handle: RePEc:nat:nature:v:431:y:2004:i:7006:d:10.1038_nature02895
    DOI: 10.1038/nature02895
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    Cited by:

    1. Peter Mayrhofer & Markus R. Anneser & Kristina Schira & Carina A. Sommer & Ina Theobald & Martin Schlapschy & Stefan Achatz & Arne Skerra, 2024. "Protein purification with light via a genetically encoded azobenzene side chain," Nature Communications, Nature, vol. 15(1), pages 1-18, December.

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