MUTATIONS IN THE YEAST MYB-LIKE PROTEIN BAS1P RESULTING IN DISCRIMINATION BETWEEN PROMOTERS IN-VIVO BUT NOT IN-VITRO

Bas1p is a yeast transcription factor that activates expression of pur ine and histidine biosynthesis genes in response to extracellular puri ne limitation. The N-terminal part of Bas1p contains an Myb-like DNA b inding domain composed of three tryptophan-rich imperfect repeats. We show that mutating the conserved tryptophan residues in the DNA bindin g domain of Bas1p severely impairs in vivo activation of target genes and in vitro DNA binding of Bas1p. We also found that two mutations (H 34L and W42A) in the first repeat make Bas1p discriminate between prom oters 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 p romoters in vitro, suggesting that the mutations affect the interactio n 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 re gulation of Bas1p activity. Finally, using a green fluorescence protei n (GFP)-Bas1p fusion, we establish the strict nuclear localization of Bas1p and show that it is not affected by extracellular adenine.