engleski

Complex Role of Pho2 in the Activation of Yeast PHO5

Two transcriptional factors, the bHLH protein Pho4 and homeodomain protein Pho2, are required for transcriptional activation of the PHO5 promoter in S. cerevisiae. There are two essential Pho4 binding sites at the PHO5 promoter corresponding to regulatory elements UASp1 and UASp2. Multiple Pho2 binding sites, closely adjacent or overlapping Pho4 binding sites have been recently identified, and we have demonstrated that two proteins bind cooperatively to both UASp elements in vitro. We have now investigated the importance of cooperative interactions of two proteins for the promoter activation in vivo. A mutation of the high affinity Pho2 site overlapping Pho4 site at UASp1 leads to a significant loss of cooperative DNA binding, which correlates with 3-fold decrease of the promoter activity. Separate mutations of the Pho2 sites adjacent to UASp2 result in smaller decrease in transcriptional activity, although cooperative DNA binding was fully eliminated. However, by combining these mutations, the PHO5 promoter was almost completely inactivated, demonstrating functional importance of mapped Pho2 binding sites for the promoter activity. Use of Pho4 derivative with an internal deletion of a domain which interacts in the two-hybrid system with Pho2, leads to a loss of cooperative DNA binding and dramatic loss of the promoter activity. However, in a heterologous promoter constructs UASp2 activates strongly with this derivative, while UASp1 is fully inactive, showing a striking difference in requirements of the two UASp elements for cooperative Pho2-Pho4 interactions. From in vivo footprint experiments and activity measurements with a promoter variant containing two UASp2 elements we conclude that at UASp2 Pho2 is mainly required for the ability of Pho4 to transactivate, while cooperative Pho2-Pho4 interactions are critical for binding of Pho4 to UASp1. These results demonstrate importance of the complex protein-protein and protein-DNA interactions that lead to transcriptional activation and chromatin remodeling of the PHO5 gene.

Saccharomyces cerevisiae; PHO5; Pho2; Pho4; transcriptional regulation

nije evidentirano