Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein.
The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site 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 excess of nonspecific carrier DNA was purified. Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DNA affinity chromatography. In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively. Methylation interference and missing-nucleoside studies demonstrated that the two proteins bind at adjacent sites, with each protein making unique DNA contacts. SWI5 and GRF10 interactions were not detected in the absence of DNA. The role of cooperative DNA binding in determining promoter specificity of eukaryotic transcription factors is discussed.
Brazas
R M
RM
Department of Cellular, Viral, and Molecular Biology, University of Utah Medical Center, Salt Lake City 84132.
Stillman
D J
DJ
D000494:Allosteric Regulation; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D005800:Genes, Fungal; D018398:Homeodomain Proteins; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000302:isolation & purification*; D014157:Transcription Factors / Q000378:metabolism*; D016335:Zinc Fingers
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D018398:Homeodomain Proteins; D009838:Oligodeoxyribonucleotides; C051611:PHO2 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; C080218:SWI5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors