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

•A chimera of tRNASer and tRNASec, tRNAUTuX, binds EF-Tu to insert Sec at UAG codons.•tRNAUTuX was used for complete, high fidelity Sec insertion.•We show in vitro selenoprotein synthesis, compatible with wild-type and synthetic tRNA.•Sense codons were recoded in vitro in the presence of SelB.•Formate dehydrogenase activity demonstrates in vitro selenoenzyme synthesis. 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.