HAP1 positive control mutants specific for one of two binding sites.
The expression of the yeast CYC1 and CYC7 genes is controlled by the HAP1 activator. A GAL4-like zinc finger (residues 1-148) specifies binding to the dissimilar sites UAS1 (of CYC1) and CYC7, and an acidic domain (residues 1307-1483) is essential for activation of transcription. To analyze how HAP1 binds to UAS1 and CYC7, we performed saturation mutagenesis of the DNA-binding domain and recovered mutants with altered activity. Class 1 mutants had a reduced activity at both UAS1 and CYC7, and class 2 mutants selectively eliminated activity at CYC7. Surprisingly, several mutants of both classes exhibited wild-type DNA binding, indicating that they were specifically defective in activation. These positive control (PC) mutants alter residues that bracket the zinc finger. We explain these mutants in a model involving cofactor proteins that bind UAS1 and CYC7 along with HAP1. The existence of PC mutants that only affect activity at CYC7 raises the possibility that different cofactors may exist for UAS1 and CYC7.
Turcotte
B
B
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Guarente
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003574:Cytochrome c Group / Q000235:genetics; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D016335:Zinc Fingers / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D003574:Cytochrome c Group; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C058452:HAP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D001616:beta-Galactosidase