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.•Forma...

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Bibliographic Details
Published in:	FEBS letters Vol. 589; no. 17; pp. 2194 - 2199
Main Authors:	Miller, Corwin, Bröcker, Markus J., Prat, Laure, Ip, Kevan, Chirathivat, Napon, Feiock, Alexander, Veszprémi, Miklós, Söll, Dieter
Format:	Journal Article
Language:	English
Published:	England Elsevier B.V 04-08-2015
Subjects:	Bacterial Proteins - genetics Bacterial Proteins - metabolism Base Sequence Cell-free protein synthesis Codon, Terminator - genetics Escherichia coli - genetics Escherichia coli - metabolism In vitro translation Molecular Sequence Data Mutation Non-canonical amino acid 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 Selenocysteine - metabolism Selenoprotein Selenoproteins - genetics Selenoproteins - metabolism Selenocysteine Selenoprotein Non-canonical amino acid In vitro translation Cell-free protein synthesis
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Summary:	•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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors contributed equally to this work.
ISSN:	0014-5793 1873-3468
DOI:	10.1016/j.febslet.2015.06.039