Transcription of eukaryotic genes requires an interaction between transcription factors that bind to the TATA box region, and transcriptional activators that bind to upstream activating sequences (UASs) or enhancers. Several yeast upstream transcriptional activators, such as GCN4, GAL4 and HAP1, seem to contain separate domains for binding to DNA and activating transcription. The expression of the cytochrome genes CYC1 and CYC7 is controlled by HAP1, which binds to dissimilar DNA sequences in UAS1 of CYC1 and the UAS of CYC7. HAP1 has a zinc-finger DNA-binding domain between amino-acid residues 1 and 148, and a highly acidic C-terminal activation domain between residues 1,308 and 1,483 (ref. 10). A mutant allele of the HAP1 gene, HAP1-18, leads to a change in Ser 63 to Arg 63, immediately adjacent to the zinc finger in the DNA-binding domain. The HAP1-18 mutation specifically abolishes the ability of the protein to bind to UAS1, but greatly increases the ability of the protein to activate transcription of CYC7. We now report that this increase in activation is mediated solely by the CYC7 UAS and the HAP1-18 protein, and also, that it is not caused by an altered binding affinity of the protein for the CYC7 UAS. Furthermore, even by substituting other amino acids at position 63 and over-expressing the resulting derivatives in vivo we were unable to increase activity at the UAS of CYC7 to the level obtained with HAP1-18. This rules out the possibility that the HAP1-18 mutation increases transcriptional activation by abolishing competition by UAS1 and UAS1-like sites for the protein. We thus conclude that HAP1-18 is a better activator of transcription than the wild-type protein when bound to the UAS of CYC7. Moreover, our findings indicate that in addition to the acidic activation domain, the zinc-finger DNA-binding domain participates directly in the activation of transcription.

• PMID: 2509943
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Journal Article | Research Support, U.S. Gov't, P.H.S.

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Kim KS, Guarente L

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