DETERMINING THE REQUIREMENTS FOR COOPERATIVE DNA-BINDING BY SWI5P ANDPHO2P (GRF10P BAS2P) AT THE HO PROMOTER/

SW15 encodes a zinc finger DNA binding protein required for the transc ription of the Saccharonyces cerevisiae HO gene, and PHO2 encodes a ho meodomain DNA binding protein. In vitro biochemical studies using puri fied Swi5p and Pho2p proteins have demonstrated that Swi5p and Pho2p b ind cooperatively to the HO promoter. in this report we investigate th e regions of the Swi5p and Pho2p proteins required for cooperative DNA binding. The analysis of each protein gives a similar result: the zin c finger or homeodomain DNA binding domains are each sufficient for in vitro DNA binding, but additional regions of each protein are require d for cooperative DNA binding. In vitro and in vivo experiments were c onducted with promoters with altered spacing between the Pho2p and Swi 5p binding sites. Mutations that increased the distance between the tw o binding sites had minimal effects on either in vitro cooperative DNA binding or in vivo upstream activating sequence activity. These obser vations suggest that the interaction domains of Swi5p and Pho2p are fl exible and can tolerate an increase in distance between the two bindin g sites. The mechanism of the cooperative DNA binding by Swi5p and Pho 2p is discussed.