NEGATIVE TRANSCRIPTIONAL REGULATION OF PHO81 EXPRESSION IN SACCHAROMYCES-CEREVISIAE

The PH081 gene product functions as an inhibitor of the cyclin-cyclin- dependent kinase pair, Pho80-Pho85, and is required for derepression o f acid phosphatase-encoding gene (PHO5) expression. PHO81 is the only known regulator of this system whose transcriptional expression is reg ulated by the level of inorganic phosphate. This effect is mediated by the gene products of the PHO4 and PHO2 (BAS2, GRF10) genes which act as transcription factors, Fine structural analysis of the PH081 promot er region has revealed the existence of a negative regulatory sequence (NRS). That is, removal of this element causes an approx. 4-fold incr ease in PHO81 expression. The NRS functions in either orientation, but only when located downstream from the activation sequences. Interesti ngly, this element shows significant homology to a sequence present in the promoter of the PHO8 gene, encoding a phosphate-repressible alkal ine phosphatase. An electrophoretic mobility shift assay (EMSA) using whole-cell extracts and a NRS-containing DNA fragment detects a protei n which specifically binds to this element.