Eukaryotic selenocysteine incorporation follows a nonprocessive mechanism that competes with translational termination

The synthesis of eukaryotic selenoproteins involves the recoding of an inte rnal UGA codon as a site for selenocysteine incorporation. This recoding ev ent is directed by a selenocysteine insertion sequence in the 3'-untranslat ed region. Because UGA also functions as a signal for peptidyl-tRNA hydroly sis, we have investigated how the rates of translational termination and se lenocysteine incorporation relate to cia-acting elements in the mRNA as wel l as to trans-acting factors in the cytoplasm, We used cia-elements from th e phospholipid glutathione peroxidase gene as the basis for this work becau se of its relatively high efficiency of selenocysteine incorporation. The l ast two codons preceding the UGA were found to exert a far greater influenc e on selenocysteine incorporation than nucleotides downstream of it, The ef ficiency of selenocysteine incorporation was generally much less than 100% but could be partially enhanced by concomitant overexpression of the tRNA(S ec) gene. The combination of two or three UGA codons in one reading frame l ed to a dramatic reduction in the yield of full-length protein. It is there fore unlikely that multiple incorporations of selenocysteine are processive with respect to the mode of action of the ribosomal complex binding to the UGA site. These observations are discussed in terms of the mechanism of se lenoprotein synthesis and its ability to compete with termination at UGA co dons.