Promoter analysis of the PHO81 gene encoding a 134 kDa protein bearing ankyrin repeats in the phosphatase regulon of Saccharomyces cerevisiae

Abstract

The PHO81 gene encoding one of the regulators of the phosphatase regulon in Saccharomyces cerevisiae was mapped 9.8 centimorgans distal from the ser2 locus on the right arm of chromosome VII. Determination of the nucleotide sequence of cloned PHO81 DNA revealed a 3537 bp open reading frame encoding a 134 kDa protein. This protein has six repeats of a 33-amino acid sequence homologous to the ankyrin repeat and an asparagine-rich region. Transcription of PHO81 is activated by Pho4 protein in cooperation with Pho2 (i.e., Bas2/Grf10) protein under the influence of the inorganic phosphate (Pi) concentration in the medium, through the PHO regulatory system. Major transcription initiation sites of PHO81, determined by primer extension analysis, are at nucleotide positions -66 and -65 relative to the ATG codon. Deletion analysis showed that a 95 bp region from nucleotide position -385 to -291 is essential for response to the Pi signals. Purified Pho4 protein protected a 19 bp region (positions -350 to -332) in the 95 bp fragment from DNase I digestion in vitro and the protected region includes the core sequence 5'-CACGTG-3', which is also observed in other genes of phosphate metabolism.