Interaction of the Yeast PH02 Protein or Its Mutants with the PHO5 UAS in vitro

The GST gene fusion system was used to express PHO2 gene and its mutants in E. coli Gel retardation assays showed that PHO2 fusion protein can bind to the upstream activation sequence (UAS) of the acid phosphatase gene PHO5. The homeodomain of PHO2 protein has such structure as alpha-helix 2-beta-turn-alpha-helix 3, which acts as the DNA binding domain of the transcriptional factor. The Mutation of lle 123 to Pro in helix 3 or the insertion of 4 amino acids (PDPD) between 112 and 113 in alpha-helix 2 led to the complete loss of DNA-binding activity of PHO2, while the mutation of Pro 117 to Ala in beta-turn did not affect the binding activity significantly. Deletions of the PHO80 homologous region, acidic region or C-terminal 132 residues had no great effect on the DNA-binding activity, although these mutants had lost the ability to activate PHO5 expression in vivo.