Journal
FEBS LETTERS
Volume 589, Issue 17, Pages 2194-2199Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.febslet.2015.06.039
Keywords
Selenoprotein; Selenocysteine; Non-canonical amino acid; Cell-free protein synthesis; In vitro translation
Funding
- National Institute for General Medical Sciences [GM22854]
- Defense Advanced Research Projects Agency [N66001-12-C-4020, N66001-12-C-4211]
- Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the Department of Energy [DE-FG02-98ER20311]
- Alexander von Humboldt Foundation (Bonn, Germany)
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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. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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