SELENOCYSTEINE INSERTION OR TERMINATION - FACTORS AFFECTING UGA CODONFATE AND COMPLEMENTARY ANTICODON-CODON MUTATIONS

Translation of UGA as selenocysteine instead of termination occurs in numerous proteins, and the process of recoding UGA requires specific s ignals in the corresponding mRNAs. In eukaryotes, stem - loops in the 3' untranslated region of the mRNAs confer this function. Despite the presence of these signals, selenocysteine incorporation is inefficient . To investigate the reason for this, we examined the effects of the a mount of deiodinase cDNA on UGA readthrough in transfected cells, quan titating the full-length and UGA terminated products by Western blotti ng. The gene for the selenocysteine-specific tRNA was also cotransfect ed to determine if it was limiting. We find that the concentrations of both the selenoprotein DNA and the tRNA affect the ratio of selenocys teine incorporation to termination. Selenium depletion was also found to decrease readthrough. The fact that the truncated peptide is synthe sized intracellularly demonstrates unequivocally that UGA can serve as both a stop and a selenocysteine codon in a single mRNA. Mutation of UGA to UAA (stop) or UUA (leucine) in the deiodinase mRNA abolishes de iodinase activity; but activity is partially restored when selenocyste ine tRNAs containing complementary mutations are cotransfected. Thus, UGA is not essential for selenocysteine incorporation in mammalian cel ls, provided that codon:anticodon complementarity is maintained.