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Regions of GAL4 critical for binding to a promoter in vivo revealed by a visual DNA‐binding analysis

Binding of transcriptional activators to specific sites on DNA, mediated by their DNA‐binding domain, is a key regulatory point in transcriptional regulation. With a GFP‐based microscopic assay, we investigated how the prototypical activator GAL4 effectively binds to a promoter in living yeast cells. We show that GAL4 relies on a previously unrevealed mechanism involving ‘DNA‐binding enhancers’ (DBEs), the regions of GAL4 that assist DNA‐binding domain association with DNA. GAL4 contains two DBEs, one, but not the other, physically overlapping the principal transcriptional activation domain. Either of the DBEs, however, can function independently of transcriptional activation, indicating a discrete mechanism responsible for DNA‐binding enhancement. The effect of DBEs, while not limited to natural target promoters, is still not universal and can be profoundly affected by the binding‐site context. The GAL4 DBEs can also enhance promoter binding of an unrelated DNA‐binding domain, and possibly represent a new modular functional unit responsible for effective binding of diverse regulatory factors to DNA in vivo.