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 (3′ UTR), Sec-tRNASec, the Sec-specific elongation factor eEFSec, and SECIS binding protein 2 (SBP2). 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-tRNASec 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.

Background: Selenocysteine (Sec) incorporation requires the function of a unique translation elongation factor, eEFSec. Results: The novel Domain IV of eEFSec is required for at least three functions. Conclusion: Domain IV of eEFSec is the key site for dictating specificity in the conversion of the UGA stop codon into a Sec codon. Significance: Understanding elongation factor function is critical to deciphering the mechanism of Sec incorporation.