The human p53 negative regulatory domain mediates inhibition of reporter gene transactivation in yeast lacking thioredoxin reductase.
Stimulation of target gene transcription by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. LexA/p53 fusion proteins were used to study the basis for thioredoxin reductase dependence. A fusion protein containing all 393 of the residues of p53 efficiently and specifically stimulated transcription of a LexOP-LacZ reporter gene in wild-type yeast but was several-fold less effective in delta trr1 yeast lacking the thioredoxin reductase gene. Thus, even when p53 was tethered to a reporter gene by a heterologous DNA-binding domain, reporter gene transactivation remained dependent on thioredoxin reductase. A fusion protein containing only the activation domain of p53 stimulated reporter gene transcription equally in wild-type and delta trr1 cells, suggesting that p53 residues downstream from the activation domain created the requirement for thioredoxin reductase. Experiments using additional LexA/p53 truncation mutations indicated that the p53 negative regulatory domain, rather than the DNA-binding or oligomerization domains, created the requirement for thioredoxin reductase. The fusion protein results suggested that, under oxidative conditions, the negative regulatory domain inhibited the ability of DNA-bound p53 to stimulate transcription. However, deletion of the negative regulatory domain did not alleviate the requirement of non-LexA-containing p53 for thioredoxin reductase. The results, thus, suggest that oxidative conditions inhibit both DNA binding and transactivation by p53, and that inhibition of the latter requires the negative regulatory domain.
Department of Biochemistry and Biophysics and the Center for Gene Research and Biotechnology, Oregon State University, Corvallis 97331, USA. merrillg@ucs.orst.edu
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D017930:Genes, Reporter; D016158:Genes, p53; D006801:Humans; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D011993:Recombinant Fusion Proteins; D013880:Thioredoxin-Disulfide Reductase; D001616:beta-Galactosidase