Mutations in the yeast Myb-like protein Bas1p resulting in discrimination between promoters in vivo but notin vitro.
Bas1p is a yeast transcription factor that activates expression of purine and histidine biosynthesis genes in response to extracellular purine limitation. The N-terminal part of Bas1p contains an Myb-like DNA binding domain composed of three tryptophan-rich imperfect repeats. We show that mutating the conserved tryptophan residues in the DNA binding domain of Bas1p severely impairs in vivo activation of target genes and in vitro DNA binding of Bas1p. We also found that two mutations (H34L and W42A) in the first repeat make Bas1p discriminate between promoters in vivo . These two BAS1 mutants are able to activate expression of an HIS4-lacZ fusion but not that of ADE1-lacZ or ADE17-lacZ fusions. Surprisingly, these mutant proteins bind equally well to the three promoters in vitro , suggesting that the mutations affect the interaction of Bas1p with some promoter-specific factor(s) in vivo . By mutating a potential nucleotide binding site in the DNA binding domain of Bas1p, we also show that this motif does not play a major role in purine regulation of Bas1p activity. Finally, using a green fluorescence protein (GFP)-Bas1p fusion, we establish the strict nuclear localization of Bas1p and show that it is not affected by extracellular adenine.
Institut de Biochimie et Génétique Cellulaires, CNRS UPR9026, 1 rue Camille Saint-Saëns, F-33077 Bordeaux Cedex, France.
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B
Pinson
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Sagot
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Borne
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Gabrielsen
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Daignan-Fornier
D000225:Adenine / Q000096:biosynthesis / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D002451:Cell Compartmentation / Q000187:drug effects; D002467:Cell Nucleus; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D017930:Genes, Reporter; D006639:Histidine / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D012091:Repetitive Sequences, Nucleic Acid; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
C061767:BAS1 protein, S cerevisiae; D005656:Fungal Proteins; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D006639:Histidine; D014364:Tryptophan; D000225:Adenine