A synthetic tRNA for EF-Tu mediated selenocysteine incorporation in vivo and in vitro

FEBS Lett. 2015 Aug 4;589(17):2194-9. doi: 10.1016/j.febslet.2015.06.039. Epub 2015 Jul 6.

Abstract

Incorporation of selenocysteine (Sec) in bacteria requires a UGA codon that is reassigned to Sec by the Sec-specific elongation factor SelB and a conserved mRNA motif (SECIS element). These requirements severely restrict the engineering of selenoproteins. Earlier, a synthetic tRNASec was reported that allowed canonical Sec incorporation by EF-Tu; however, serine misincorporation limited its scope. We report a superior tRNASec variant (tRNAUTuX) that facilitates EF-Tu dependent stoichiometric Sec insertion in response to UAG both in vivo in Escherichia coli and in vitro in a cellfree protein synthesis system. We also demonstrate recoding of several sense codons in a SelB supplemented cell-free system. These advances in Sec incorporation will aid rational design and directed evolution of selenoproteins.

Keywords: Cell-free protein synthesis; In vitro translation; Non-canonical amino acid; Selenocysteine; Selenoprotein.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Codon, Terminator / genetics
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Molecular Sequence Data
  • Mutation
  • Nucleic Acid Conformation
  • Peptide Elongation Factor Tu / genetics
  • Peptide Elongation Factor Tu / metabolism*
  • Peptide Elongation Factors / genetics
  • Peptide Elongation Factors / metabolism
  • Protein Biosynthesis*
  • RNA, Transfer, Amino Acid-Specific / chemistry
  • RNA, Transfer, Amino Acid-Specific / genetics
  • RNA, Transfer, Amino Acid-Specific / metabolism*
  • RNA, Transfer, Amino Acyl / chemistry
  • RNA, Transfer, Amino Acyl / genetics
  • RNA, Transfer, Amino Acyl / metabolism
  • Selenocysteine / metabolism*
  • Selenoproteins / genetics
  • Selenoproteins / metabolism

Substances

  • Bacterial Proteins
  • Codon, Terminator
  • Peptide Elongation Factors
  • RNA, Transfer, Amino Acid-Specific
  • RNA, Transfer, Amino Acyl
  • SelB protein, Bacteria
  • Selenoproteins
  • selenocysteinyl-tRNA
  • tRNA, selenocysteine-
  • Selenocysteine
  • Peptide Elongation Factor Tu