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The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions
Abstract:
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.
Insights
The repressible acid phosphatase gene PHO5 in Saccharomyces cerevisiae requires PHO2 and PHO4 proteins for expression. Both proteins bind to the PHO5 promoter, with PHO4 binding at two sites and PHO2 binding between them.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Gene Regulation
Background:
- The PHO5 gene in Saccharomyces cerevisiae encodes a repressible acid phosphatase.
- Its expression is regulated by the positively acting proteins PHO2 and PHO4.
- Mutations in PHO2 or PHO4 prevent PHO5 gene derepression under low-phosphate conditions.
Purpose of the Study:
- To investigate the in vitro binding of PHO2 and PHO4 proteins to the PHO5 promoter.
- To identify the specific binding sites for PHO2 and PHO4 on the PHO5 promoter.
Main Methods:
- Gel retardation assays with promoter deletions.
- DNase I footprinting analysis.
- Exonuclease III footprinting experiments.
Main Results:
- Both PHO2 and PHO4 proteins bind specifically to the PHO5 promoter in vitro.
- PHO4 binds to two distinct regions: UASp1 (-347 to -373) and UASp2 (-239 to -262).
- PHO2 binds to a region located between the two PHO4-binding sites (-277 to -296).
Conclusions:
- PHO2 and PHO4 directly interact with the PHO5 promoter.
- The identified binding sites are crucial for the regulation of PHO5 gene expression.
- This study elucidates the molecular mechanism of PHO5 gene activation by PHO2 and PHO4.