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Biochem Biophys Res Commun. 2008 Aug 1;372(3):480-5. doi: 10.1016/j.bbrc.2008.05.078. Epub 2008 May 27.

Phosphoserine aminoacylation of tRNA bearing an unnatural base anticodon.

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Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.


An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a K(m)=47.1muM and a k(cat)=0.151s(-1), which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNA(Cys) with the GCA anticodon (26.9muM and 0.111s(-1)). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.

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