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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Jul 06, 2015
- Source ID
- 10.1016/j.febslet.2015.06.039
Entities
People
- Alexander Feiock
- Corwin Miller
- Dieter Söll
- Kevan Ip
- Laure Prat
- Markus J. Bröcker
- Miklós Veszprémi
- Napon Chirathivat
Organizations
- Defense Advanced Research Projects Agency
- National Institute of General Medical Sciences