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A synthetic tRNA for EF‐Tu mediated selenocysteine incorporation in vivo and in vitro
Author(s) -
Miller Corwin,
Bröcker Markus J.,
Prat Laure,
Ip Kevan,
Chirathivat Napon,
Feiock Alexander,
Veszprémi Miklós,
Söll Dieter
Publication year - 2015
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2015.06.039
Subject(s) - selenocysteine , transfer rna , biochemistry , biology , protein biosynthesis , serine , escherichia coli , stop codon , elongation factor , amino acid , in vitro , chemistry , ribosome , rna , enzyme , gene , cysteine
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 tRNA Sec was reported that allowed canonical Sec incorporation by EF‐Tu; however, serine misincorporation limited its scope. We report a superior tRNA Sec variant (tRNA UTuX ) 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.