The homeodomain protein Pho2p binds at an A/T-rich segment flanking the binding site of the basic-helix-loop-helix protein Pho4p in the yeast PHO promoters.
Transcription of the genomic PHO5, PHO81 and PHO84 genes of the PHO regulon requires Pho4p and Pho2p activity, whereas transcription of PHO8 is directed by Pho4p alone. Pho4p binds to two 9-bp motifs, 5'-GCACGTGGG-3' (type 1. e.g. UASp2 of PHO5 and site D of PHO84) and 5'-GCACGTTTT-3' (type 2, e.g. UASp1 of PHO5 and site E of PHO84) in the PHO promoter. Experiments were performed to evaluate the ability of these 9-bp motifs to function as upstream activation sites (UASs) by insertion of various 36-bp fragments bearing the 9-bp motif in a CYC1-lacZ fusion gene. No expression of the lacZ gene was detected with the 36-bp fragment bearing UASp2 of PHO5, whereas similar 36-bp fragments bearing UASp1 of PHO5 and sites D and E of PHO84 showed UAS activity in response to Pi concentration in the medium and to the pho2 mutation. The Pho2p-responsive UASs are flanked by one or two copies of an A/T-rich segment, whereas UASp2 is not. Gel retardation and competition experiments performed using a T7-Pho2p-His chimeric protein showed that Pho2p binds to the 36-bp fragments bearing A/T-rich segment(s) but not appreciably to the 36-bp fragments not bearing such segment(s). Thus, the A/T segments flanking the PHO UASs are Pho2p binding sites and play an important role in PHO regulation.
Magbanua
J P
JP
Department of Biotechnology, Faculty of Engineering, Osaka University, Japan.
Fujisawa
K
K
Ogawa
N
N
Oshima
Y
Y
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D018257:Helix-Loop-Helix Motifs / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors
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