RESIDUES IN THE SWI5 ZINC-FINGER PROTEIN THAT MEDIATE COOPERATIVE DNA-BINDING WITH THE PHO2 HOMEODOMAIN PROTEIN

The Swi5 zinc finger and the Pho2 homeodomain DNA-binding proteins bin d cooperatively to the HO promoter. Pho2 (also known as Bas2 or Grf10) activates transcription of diverse genes, acting with multiple distin ct DNA-binding proteins, We have performed a genetic screen to identif y amino acid residues in Swi5 that are required for synergistic transc riptional activation of a reporter construct in vivo. Nine unique amin o acid substitutions within a 24-amino-acid region of Swi5, upstream o f the DNA-binding domain, reduce expression of promoters that require both Swi5 and Pho2 for activation. In vitro DNA binding experiments sh ow that the mutant Swi5 proteins bind DNA normally, but some mutant Sw i5 proteins (resulting from SWI5 mutations) show reduced cooperative DNA binding with Pho2. In vivo experiments show that these SWI5 mutat ions sharply reduce expression of promoters that require both SWI5 and PHO2, while expression of promoters that require SWI5 but are PHO2 in dependent is largely unaffected. This suggests that these SWI5 mutati ons do not affect the ability of Swi5 to bind DNA or activate transcri ption but specifically affect the region of Swi5 required for interact ion with Pho2. Two-hybrid experiments show that amino acids 471 to 513 of Swi5 are necessary and sufficient for interaction with Pho2 and th at the SWI5 point mutations cause a severe reduction in this two-hybr id interaction. Analysis of promoter activation by these mutants sugge sts that this small region of Swi5 has at least two distinct functions , conferring specificity for activation of the HO promoter and for int eraction with Pho2.