Determining the requirements for cooperative DNA binding by Swi5p and Pho2p (Grf10p/Bas2p) at the HO promoter

SW15 encodes a zinc finger DNA binding protein required for the transcription of the Saccharomyces cerevisiae HO gene, and PHO2 encodes a homeodomain DNA binding protein. In vitro biochemical studies using purified Swi5p and Pho2p proteins have demonstrated that Swi5p and Pho2p bind cooperatively to the HO promoter. In this report we investigate the regions of the Swi5p and Pho2p proteins required for cooperative DNA binding. The analysis of each protein gives a similar result: the zinc finger or homeodomain DNA binding domains are each sufficient for in vitro DNA binding, but additional regions of each protein are required for cooperative DNA binding. In vitro and in vivo experiments were conducted with promoters with altered spacing between the Pho2p and Swi5p binding sites. Mutations that increased the distance between the two binding sites had minimal effects on either in vitro cooperative DNA binding or in vivo upstream activating sequence activity. These observations suggest that the interaction domains of Swi5p and Pho2p are flexible and can tolerate an increase in distance between the two binding sites. The mechanism of the cooperative DNA binding by Swi5p and Pho2p is discussed.