Internal deletions in the yeast transcriptional activator HAP1 have opposite effects at two sequence elements.
In this report we study the effects of internal deletions of the yeast transcriptional activator HAP1 (CYP1) on activity at two dissimilar DNA binding sites, upstream activation sequence 1 (UAS1) of CYC1 (iso-1-cytochrome c) and CYC7 (iso-2-cytochrome c). These deletions remove up to 1061 amino acids of the 1483-residue protein and bring the carboxyl-terminal acidic activation domain closer to the amino-terminal DNA-binding domain. Surprisingly, the deletions have opposite effects at the two sites; activity at UAS1 increases with deletion size, while activity at CYC7 decreases. The mutant with the largest deletion, mini-HAP1, has no measurable activity at CYC7 but binds normally to the site in vitro. In contrast, a protein with the DNA-binding domain of HAP1 fused to the acidic activation domain of GAL4 is active at both UAS1 and CYC7. These findings are discussed in the context of two models that suggest how the DNA sequence can alter the activity of the bound HAP1. In a separate experiment, we generate a mutation in the DNA-binding domain of HAP1 that requires the addition of zinc for binding to either UAS1 or CYC7 in vitro. This finding shows that a zinc finger anchors DNA binding to both types of HAP1 sites.
Kim
K S
KS
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Pfeifer
K
K
Powell
L
L
Guarente
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D002872:Chromosome Deletion; D003574:Cytochrome c Group / Q000235:genetics*; D045304:Cytochromes c; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D008667:Metalloproteins / Q000235:genetics; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C058452:HAP1 protein, S cerevisiae; D008667:Metalloproteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C032139:iso-2-cytochrome C; D045304:Cytochromes c