title: A synthetic tRNA for EF-Tu mediated selenocysteine incorporation in vivo and in vitro.
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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA. Electronic address: corwin.miller@yale.edu.
authors.forename: Corwin
authors.initials: C
authors.lastname: Miller
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Markus J
authors.initials: MJ
authors.lastname: Bröcker
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Laure
authors.initials: L
authors.lastname: Prat
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Kevan
authors.initials: K
authors.lastname: Ip
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Napon
authors.initials: N
authors.lastname: Chirathivat
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Feiock
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Miklós
authors.initials: M
authors.lastname: Veszprémi
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA; Department of Chemistry, Yale University, New Haven, CT 06520, USA. Electronic address: dieter.soll@yale.edu.
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018388:Codon, Terminator / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D012345:RNA, Transfer, Amino Acid-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017279:Selenocysteine / Q000378:metabolism*; D051140:Selenoproteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018388:Codon, Terminator; D010445:Peptide Elongation Factors; D012345:RNA, Transfer, Amino Acid-Specific; D012346:RNA, Transfer, Amino Acyl; C061878:SelB protein, Bacteria; D051140:Selenoproteins; C112705:selenocysteinyl-tRNA; C036928:tRNA, selenocysteine-; D017279:Selenocysteine; D010444:Peptide Elongation Factor Tu
keywords: Cell-free protein synthesis; In vitro translation; Non-canonical amino acid; Selenocysteine; Selenoprotein