Interplay between termination and translation machinery in eukaryotic selenoprotein synthesis.
Termination of translation in eukaryotes is catalyzed by eRF1, the stop codon recognition factor, and eRF3, an eRF1 and ribosome-dependent GTPase. In selenoprotein mRNAs, UGA codons, which typically specify termination, serve an alternate function as sense codons. Selenocysteine incorporation involves a unique tRNA with an anticodon complementary to UGA, a unique elongation factor specific for this tRNA, and cis-acting secondary structures in selenoprotein mRNAs, termed SECIS elements. To gain insight into the interplay between the selenocysteine insertion and termination machinery, we investigated the effects of overexpressing eRF1 and eRF3, and of altering UGA codon context, on the efficiency of selenoprotein synthesis in a transient transfection system. Overexpression of eRF1 does not increase termination at naturally occurring selenocysteine codons. Surprisingly, selenocysteine incorporation is enhanced. Overexpression of eRF3 did not affect incorporation efficiency. Coexpression of both factors reproduced the effects with eRF1 alone. Finally, we show that the nucleotide context immediately upstream and downstream of the UGA codon significantly affects termination to incorporation ratios and the response to eRF overexpression. Implications for the mechanisms of selenocysteine incorporation and termination are discussed.
Grundner-Culemann
E
E
Thyroid Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Martin
G W
GW
Tujebajeva
R
R
Harney
J W
JW
Berry
M J
MJ
D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003062:Codon / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007453:Iodide Peroxidase / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010443:Peptide Chain Termination, Translational / Q000235:genetics*; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000235:genetics*; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012643:Selenium / Q000378:metabolism; D017279:Selenocysteine / Q000235:genetics / Q000378:metabolism*; D051140:Selenoproteins; D014162:Transfection
D003062:Codon; C489002:ETF1 protein, human; D010454:Peptide Termination Factors; D011506:Proteins; D012333:RNA, Messenger; D051140:Selenoproteins; C087971:peptide-chain-release factor 3; D017279:Selenocysteine; D007453:Iodide Peroxidase; D012643:Selenium