Mj. Berry et al., SELENOCYSTEINE INSERTION OR TERMINATION - FACTORS AFFECTING UGA CODONFATE AND COMPLEMENTARY ANTICODON-CODON MUTATIONS, Nucleic acids research, 22(18), 1994, pp. 3753-3759
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.