The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions.
The repressible acid phosphatase gene PHO5 of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHO5 gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHO5 promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PHO4 binding. Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASp1) and the other located at -239 to -262 (UASp2). Exonuclease III footprint experiments revealed stops at -349 and -368 (UASp1) as well as at -245 and -260 (UASp2). Gel retardation assays with the PHO2 protein revealed a binding region that lay between the two PHO4-binding sites. DNase I footprint analysis suggested a PHO2-binding site covering the region between -277 and -296.
Vogel
K
K
Biotechnology Department, CIBA-GEIGY AG, Basel, Switzerland.
Hörz
W
W
Hinnen
A
A
D000135:Acid Phosphatase / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005800:Genes, Fungal; D018398:Homeodomain Proteins; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D018398:Homeodomain Proteins; C051611:PHO2 protein, S cerevisiae; C060292:PHO4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000135:Acid Phosphatase