ROLE OF NEGATIVE REGULATION IN PROMOTER SPECIFICITY OF THE HOMOLOGOUSTRANSCRIPTIONAL ACTIVATORS ACE2P AND SWI5P

The Ace2p and Swi5p zinc finger proteins have nearly identical DNA-bin ding domains, yet in vivo they activate transcription of different gen es, CTS1 and HO. We now demonstrate that Ace2p and Swi5p recognize sit es in the CTS1 and HO promoters with the same affinities, raising the question of how promoter specificity is achieved by these proteins wit h similar DNA-binding domains. It has been previously shown that Swi5p binds to the HO promoter cooperatively with the Pho2p (Bas2p/Grf10p) homeodomain protein, and we now show that Ace2p does not interact with Pho2p. Analysis of CTS1 promoter fragments inserted into a heterologo us promoter identify a sequence 90 bp away from the Ace2p binding site s which is required to prevent activation by Swi5p through these bindi ng sites, These results suggest that a regulatory protein bound to the CTS1 promoter is needed to prevent Swi5p from activating CTS1 express ion. A genetic screen was conducted to identify suppressor mutations w hich allow CTS1 expression in the absence of the Ace2p activator, The nce3 mutation suppresses the ace2 defect in CTS1 expression only if th e strain contains a functional SWI5 gene, suggesting that NCE3 normall y functions to prevent Swi5p from activating CTS1, The role of negativ e regulators such as NCE3, as well as the previously described SIN5 ge ne, in determining the promoter specificity of homologous activators i s discussed.