Phosphoserine aminoacylation of tRNA bearing an unnatural base anticodon

Ryuya Fukunaga, Yoko Harada, Ichiro Hirao, Shigeyuki Yokoyama

Research output: Contribution to journalArticlepeer-review

Abstract

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 Km = 47.1 μM and a kcat = 0.151 s-1, which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNACys with the GCA anticodon (26.9 μM and 0.111 s-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.

Original languageEnglish (US)
Pages (from-to)480-485
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume372
Issue number3
DOIs
StatePublished - Aug 1 2008
Externally publishedYes

Keywords

  • Aminoacylation
  • Engineering
  • Phosphoserine
  • Synthetase
  • Unnatural base pair
  • tRNA

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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