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Direct charging of tRNACUA with pyrrolysine in vitro and in vivo


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.


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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).

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