Abstract
The promoter of the galactose-inducible yeast GCY1 gene allows high rates of basal transcription and is kept free of nucleosomes regardless of growth conditions. The general regulatory factor, Reb1p, as well as the nucleotide sequence of a single Gal4p-binding site, structurally cooperate to exclude nucleosomes from about 480 bp of DNA that spans the UAS(GAL), the Reb1p-binding site, the TATA-box, and the transcriptional initiation sites. Gal4p, which induces transcription of GCY1 about 25-fold in the presence of galactose, is not required for the alteration in chromatin configuration in the promoter upstream region since the hypersensitive site is unchanged when Gal4p is inactive or absent. As soon as either the Reb1p-binding site or the UAS(GAL) or both are mutated, nucleosomes slip into the promoter of GCY1 paralleled by a reduction of basal transcription activity to about 30% in either single mutant and to <10% in the double mutant. In the mutant of the Reb1p-binding site, induction by galactose/Gal4p restores a nucleosome-free state to an extent resembling the GCY1 wild-type promoter, showing that, in principle, activated Gal4p can exclude nucleosomes on its own. Northern blots of GCY1 transcripts confirm that Reb1p modulates basal transcription and has little influence on the galactose-induced state.
Highlights
Introduction of Genomic Mutations into thePromoter of GCY1— Deletion of the Reb1p-binding site, the point mutation of the UASGAL, as well as the double mutation of both cis-acting elements in the genomic context of the GCY1 promoter was accomplished by a two-step gene replacement
Basal expression in the absence of active Gal4p was found to be in part dependent on the general regulatory factor, Reb1p, which binds to its target site about 100 bp 5Ј of the TATA box and about 140 bp 3Ј of the UASGAL [31]
The UASGAL contributes to basal transcription of GCY1 as well, an effect that is independent of Gal4p binding
Summary
Reb1p, rDNA enhancer-binding protein; GCY1, galactose-inducible yeast gene encoding an aldo/keto reductase; RIO1, yeast gene encoding the protein kinase, Rio1p. Expression of GCY1 is induced about 25-fold by growth on galactose as carbon source due to Gal4p binding to a single UASGAL in the upstream control region [30]. We have investigated the influence of mutations in the GCY1 promoter, i.e. deletion of the Reb1p-binding site or mutation of the UASGAL, or the influence of the presence or absence of Gal4p on the nucleosomal array in the upstream control region of GCY1. Mutation of either the Reb1p-binding site or the UASGAL or both cis-acting elements results in nucleosome packaging of the respective region, indicating that Reb1p binding and the UASGAL are jointly responsible for the permanent exclusion of nucleosomes from the GCY1 promoter and exert an additive effect
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