Direct charging of tRNACUA with pyrrolysine in vitro and in vivo

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 and Joseph A. Krzycki ()
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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
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
Joseph A. Krzycki: The Ohio State University

Nature, 2004, vol. 431, issue 7006, 333-335

Abstract: 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.

Date: 2004
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DOI: 10.1038/nature02895

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