Budding yeast HAP2 is required in concert with HAP3 and HAP4 to form a heterotrimeric CCAAT-binding transcriptional activation complex at the UAS2 element of CYC1. Functional homologs of HAP2 and HAP3 have been conserved in HeLa cells where HAP2 activity corresponds to a chromatographic fraction designated CP1B. Here, we describe deletion and codon insertion mutagenesis of the Saccharomyces cerevisiae HAP2 subunit. DNA binding was assessed both in vitro and in vivo whereas subunit association and transcriptional activation were analyzed in vivo by using a bifunctional lexA-HAP2 fusion. The results indicate that the 265-amino-acid HAP2 protein contains an "essential core" of 65 amino acids, which is entirely sufficient for assembly and DNA binding of the HAP2/3/4 complex. The essential core can be divided further into a region of 44 amino acids, which is sufficient for subunit association, and a region of 21 amino acids, which is required specifically for DNA recognition. The remainder of the HAP2 protein is dispensable. The roles of HAP3 and HAP4 in the heterotrimeric complex are also assessed by using lexA fusions and a fusion of the GAL4 acidic activation domain to HAP2, which partially bypasses a deletion of HAP4 but not HAP3. These data indicate that subunits HAP2 and HAP3 are primarily responsible for site-specific DNA binding by the complex, whereas the HAP4 subunit provides the primary transcriptional activation domain. A model for the function of this regulated transcriptional activation complex is presented.

• PMID: 2123465
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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Olesen JT, Guarente L

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