IDENTIFICATION AND PURIFICATION OF A PROTEIN THAT BINDS DNA COOPERATIVELY WITH THE YEAST SW15 PROTEIN

The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein r equired for the expression of the HO gene. A protein fusion between gl utathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity comp lex in vitro with the HO promoter, which was inhibited by low concentr ations of nonspecific DNA. This result was surprising, since genetic e vidence demonstrated that SWI5 functions at the HO promoter via this s ite in vivo. A yeast factor, GRF10 (also known as PHO2 and BAS2), that promoted high-affinity binding of SWI5 in the presence of a large exc ess of nonspecific carrier DNA was purified. Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DN A affinity chromatography. In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively. Methylation interference and mi ssing-nucleoside studies demonstrated that the two proteins bind at ad jacent sites, with each protein making unique DNA contacts. SWI5 and G RF10 interactions were not detected in the absence of DNA. The role of cooperative DNA binding in determining promoter specificity of eukary otic transcription factors is discussed.