Nucleic Acids Research, 1994, Vol. 22, No. 18 3753-3759
© 1994
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Selenocysteine insertion or termination: factors affecting UGA codon fate and complementary anticodon:codon mutations
Thyroid Division, Brigham and Women's Hospital, Harvard Medical School Boston, MA 02115 1Laboratory of Experimental Carcinogenesis, National Cancer Institute, National Institutes of Health Bethesda, MD 02892, USA
*To whom correspondence should be addressed
Received May 18, 1994. Revised July 26, 1994. Accepted July 26, 1994.
Translation of UGA as selenocysteine instead of termination occurs in numerous proteins, and the process of recoding UGA requires specific signals 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 amount of deiodinase cDNA on UGA readthrough in transfected cells, quantitating the full-length and UGA terminated products by Western blotting. The gene for the selenocysteine-specific tRNA was also cotransfected to determine if it was limiting. We find that the concentrations of both the selenoprotein DNA and the tRNA affect the ratio of selenocysteine incorporation to termination. Selenium depletion was also found to decrease readthrough. The fact that the truncated peptide is synthesized 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 deiodinase activity; but activity is partially restored when selenocysteine tRNAs containing complementary mutations are cotransfected. Thus, UGA is not essential for selenocysteine incorporation in mammalian cells, provided that codon:anticodon complementarity is maintained.
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