Reconstitution of selenocysteine incorporation reveals intrinsic regulation by SECIS elements

J Mol Biol. 2013 Jul 24;425(14):2415-22. doi: 10.1016/j.jmb.2013.04.016. Epub 2013 Apr 23.


Selenoproteins are present in all three domains of life and are responsible for a major part of a cell's antioxidant defense against reactive oxygen species. Synthesis of selenoproteins requires the decoding of a UGA codon as selenocysteine (Sec) instead of translation termination. Sec is incorporated into the growing polypeptide chain during translation elongation and is known to require a set of highly specific factors: the Sec insertion sequence (SECIS) element in the 3' untranslated region, Sec-tRNA(Sec), the Sec-specific elongation factor eEFSec, and SECIS binding protein 2. Since reconstitution has not been reported, whether these factors are sufficient is unknown. Here, we report a novel in vitro translation system in which Sec incorporation has been reconstituted from purified components introduced into a Sec naive system. In addition, we developed a novel method to purify Sec-tRNA(Sec) and active eEFSec/GTP/tRNA ternary complex. We found that the known basal factors are sufficient for Sec incorporation in vitro. Using this highly manipulable system, we have also found that ribosomes from non-Sec-utilizing organisms cannot support Sec incorporation and that some SECIS elements are intrinsically less efficient than others. Having identified the essential set of factors, this work removes a significant barrier to our understanding of the mechanism of Sec incorporation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 3' Untranslated Regions
  • Animals
  • Cell Extracts
  • Cell-Free System
  • Codon, Terminator*
  • Male
  • Peptide Elongation Factors / metabolism
  • Protein Biosynthesis*
  • RNA, Messenger / genetics*
  • RNA, Transfer, Amino Acid-Specific / genetics
  • RNA, Transfer, Amino Acid-Specific / metabolism
  • RNA-Binding Proteins / metabolism
  • Rats
  • Ribosomes / metabolism
  • Selenocysteine / genetics*
  • Selenocysteine / metabolism*
  • Testis


  • 3' Untranslated Regions
  • Cell Extracts
  • Codon, Terminator
  • Peptide Elongation Factors
  • RNA, Messenger
  • RNA, Transfer, Amino Acid-Specific
  • RNA-Binding Proteins
  • tRNA, selenocysteine-
  • Selenocysteine