Overexpression of human release factor 1 alone has an antisuppressor effect in human cells.
Two eukaryotic proteins involved in translation termination have recently been characterized in in vitro experiments. Eukaryotic release factor 1 (eRF1) catalyzes the release of the polypeptide chain without any stop codon specificity. The GTP-binding protein eRF3 confers GTP dependence to the termination process and stimulates eRF1 activity. We used tRNA-mediated nonsense suppression at different stop codons in a cat reporter gene to analyze the polypeptide chain release factor activities of the human eRF1 and eRF3 proteins overexpressed in human cells. In a chloramphenicol acetyltransferase assay, we measured the competition between the suppressor tRNA and the human release factors when a stop codon was present in the ribosomal A site. Whatever the stop codon (UAA, UAG, or UGA) present in the cat open reading frame, the overexpression of human eRF1 alone markedly decreased translational readthrough by suppressor tRNA. Thus, like the procaryotic release factors RF1 and RF2 in Escherichia coli, eRF1 seems to have an intrinsic antisuppressor activity in human cells. Levels of antisuppression of overexpression of both eRF3 and eRF1 were almost the same as those of overexpression of eRF1 alone, suggesting that eRF1-eRF3 complex-mediated termination may be controlled by the expression level of eRF1. Surprisingly, when overexpressed alone, eRF3 had an inhibitory effect on cat gene expression. The results of cat mRNA stability studies suggest that eRF3 inhibits gene expression at the transcriptional level. This indicates that in vivo, eRF3 may perform other functions, including the stimulation of eRF1 activity.
Le Goff
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Biologie et Génétique du Développement, CNRS UPR 41, Université de Rennes 1, France.
Philippe
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Jean-Jean
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D015152:Blotting, Northern; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D005784:Gene Amplification; D006801:Humans; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D013489:Suppression, Genetic / Q000187:drug effects*; D014158:Transcription, Genetic
D018388:Codon, Terminator; C489002:ETF1 protein, human; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D012333:RNA, Messenger; C087971:peptide-chain-release factor 3; C489005:prfA protein, E coli; C489006:prfB protein, E coli; D012343:RNA, Transfer; D015500:Chloramphenicol O-Acetyltransferase