Transcriptional activator LEU3 of yeast. Mapping of the transcriptional activation function and significance of activation domain tryptophans.
The LEU3 protein of yeast activates a number of genes in the branched chained amino acid pathways. Native LEU3 is modulated by alpha-isopropylmalate, an intermediate in leucine biosynthesis. alpha-Isopropylmalate is needed for transcriptional activation, but not for DNA binding. We show here that the transcriptional activation function of LEU3 resides within the C-terminal 32 amino acids. An adjacent stretch of 81 residues is dispensable and apparently forms a connecting link between the activation domain and a large central region previously identified as important for modulation. The newly defined activation domain contains a cluster of three tryptophan residues, each of which was changed to alanine by site-directed mutagenesis. Surprisingly, all three Trp----Ala mutations affect modulation. One of them, Trp-864----Ala, creates a LEU3 molecule that is largely unmodulated and also is a better transcriptional activator than is wild type LEU3 ("hyperactivator"). The other two mutations (Trp-861----Ala and Trp-870----Ala) change the modulation ratio but have no effect on the maximal activation efficiency of the activator. We propose that the activation domain of LEU3 is kept silent by association with the central region of the protein and that an alpha-isopropylmalate-induced conformational change in the central region releases and thus activates the activation domain.
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
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KM
Zhou
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Kohlhaw
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015345:Oligonucleotide Probes; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014158:Transcription, Genetic; D014364:Tryptophan
D004268:DNA-Binding Proteins; C063667:LEU3 protein, S cerevisiae; D015345:Oligonucleotide Probes; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014364:Tryptophan