The yeast activator HAP1--a GAL4 family member--binds DNA in a directly repeated orientation.
The yeast transcriptional activator HAP1 contains a DNA-binding domain homologous to GAL4, PPR1, and related factors. By selecting random HAP1-binding sites, we found that HAP1, like GAL4, binds to two CGG triplets. Unlike GAL4, the CGGs in the HAP1 consensus are in a direct and not inverted orientation. Sites with inverted CGGs were not recovered, and mutations converting the direct repeat of CGGs to an inverted repeat greatly reduce HAP1-binding affinity. Also, the 6-bp spacer between the CGGs contains a consensus TA that is positioned asymmetrically. Dimethylsulfate protection patterns on six of these sites show protections and enhancements that also lie in a directly repeated orientation, suggesting that the two HAP1 DNA recognition domains of a HAP1 homodimer are oriented in a directly repeated configuration on the DNA. Moreover, substitution of the HAP1 dimerization domain with that of PPR1, which forms coiled-coils and dimerizes symmetrically, did not diminish the ability of the protein to bind selectively to a direct repeat. This result suggests that one DNA-binding domain of the HAP1 homodimer must be able to swivel 180 degrees relative to the dimerization domain to make specific contacts with the second CGG triplet. Our results present a novel example of domain swiveling in one of the two identical subunits of a homodimer to accommodate specific DNA contacts to both CGG triplets of a direct repeat.
Zhang
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L
Department of Biology, Massachusetts Institute of Technology, Cambridge 02129.
Guarente
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L
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011487:Protein Conformation; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D013463:Sulfuric Acid Esters; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C058452:HAP1 protein, S cerevisiae; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D013463:Sulfuric Acid Esters; D015534:Trans-Activators; D014157:Transcription Factors; C007482:dimethyl sulfate