title: Rap1p-binding sites in the saccharomyces cerevisiae GPD1 promoter are involved in its response to NaCl.
abstract: Mechanisms involved in transcriptional regulation of the osmotically controlled GPD1 gene in Saccharomyces cerevisiae were investigated by promoter analysis. The GPD1 gene encodes NAD(+)-dependent glycerol-3-phosphate dehydrogenase, a key enzyme in the production of the compatible solute glycerol. By analysis of promoter deletions, we identified a region at nucleotides -478 to -324, in relation to start of translation, to be of great importance for both basal activity and osmotic induction of GPD1. Electrophoretic mobility shift and DNase I footprint analyses demonstrated protein binding to parts of this region that contain three consensus sequences for Rap1p (repressor activator protein 1)-binding sites. Actual binding of Rap1p to this region was confirmed by demonstrating enhanced electrophoretic mobility of the protein-DNA complex with extracts containing an N-terminally truncated version of Rap1p. The detected Rap1p-DNA interactions were not affected by changes in the osmolarity of the growth medium. Specific inactivation of the Rap1p-binding sites by a C-to-A point mutation in the core of the consensus showed that this factor is a major determinant of GPD1 expression since mutations in all three putative binding sites for Rap1p strongly hampered osmotic induction and drastically lowered basal activity. We also show that the Rap1p-binding sites appear functionally distinct; the most distal site (core of the consensus at position -386) exhibited the highest affinity for Rap1p and was strictly required for low salt induction (< or =0.6 m NaCl), but not for the response at higher salinities (> or =0.8 m NaCl). This indicates tha different molecular mechanisms might be operational for low and high salt responses of the GPD1 promoter.
authors.affiliation: Department of Cell and Molecular Biology-Microbiology, Lundberg Laboratory, Göteborg University, Medicinaregatan 9C, S-413 90 Göteborg, Sweden.
authors.forename: P
authors.initials: P
authors.lastname: Eriksson
authors.forename: H
authors.initials: H
authors.lastname: Alipour
authors.forename: L
authors.initials: L
authors.lastname: Adler
authors.forename: A
authors.initials: A
authors.lastname: Blomberg
mesh_terms: D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+); D005993:Glycerolphosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D000089804:Shelterin Complex; D012965:Sodium Chloride; D013379:Substrate Specificity; D034501:Telomere-Binding Proteins; D014157:Transcription Factors; D014882:Water-Electrolyte Balance
chemical_list: D004268:DNA-Binding Proteins; C440829:RAP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins; D014157:Transcription Factors; D012965:Sodium Chloride; D005993:Glycerolphosphate Dehydrogenase; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+)