The Transcriptional Activators of the PHO Regulon, Pho4p and Pho2p, Interact Directly with Each Other and with Components of the Basal Transcription Machinery in Saccharomyces cerevisiae

Abstract

The transcriptional regulators Pho4p and Pho2p are involved in transcription of several genes in the PHO regulon of Saccharomyces cerevisiae. Genetic evidence with temperaturesensitive pho4 and pho2 mutants suggested that Pho4p and Pho2p interact with each other. Immunoprecipitation experiments showed that Pho4p and Pho2p form a complex on a 36-bp sequence bearing an upstream activation site (UAS) and protein binding assays indicated that these proteins interact directly. DNA-binding experiments with crude extracts prepared from yeast strains expressing T7-PHO4, encoding Pho4p tagged with the T7 epitope, indicated that Pho2p interacts with T7-Pho4p and enhances the binding affinity of T7-Pho4p to the UAS. Protein binding experiments also showed that both Pho4p and Pho2p could bind with the general transcription factors, TBP, TFIIB, and TFIIEβ suggesting that the Pho4p-Pho2p complex bound to the UAS activates transcription of the PHO genes by direct interaction with the general transcription factors.