engleski

Role of Pho2 in the activation of the yeast PHO5 gene

Our previous in vitro binding studies have revealed presence of multiple binding sites for Pho2 at the yeast PHO5 promoter and that Pho2 and Pho4 bind cooperatively to both UASp elements. To determine if the mapped Pho2 sites are required for activation of the PHO5 promoter, Pho2 sites located adjacent to UASp1 and UASp2 were mutated. DNA binding of Pho2 and Pho4 to the mutated promoter variants was tested in vitro by gel shift experiments, and activities of the mutated promoters were measured in vivo with the help of LacZ constructs. Mutating of Pho2 site at UASp1 completely abolished cooperativity between Pho2 and Pho4. The remaining activity of the mutated promoter (15%) corresponds to the activity of a promoter with a mutated Pho4 site at UASp1, showing that the cooperative binding of the Pho2 and Pho4 is crucial for the activity of UASp1. Analogous experiments carried out with a promoter variant containing mutated Pho2 sites adjacent to UASp2 demonstrated that this UAS element retained residual activity in the absence of Pho2 binding, but that cooperative DNA binding of the two proteins is required for full activity of UASp2. The residual activity of a promoter containing mutated Pho2 sites at both UASp1 and UASp2 (< 10%) was only slightly higher than the activity of a promoter lacking Pho4 sites altogether, confirming for the first time the central importance of the Pho2 binding sites for PHO5 promoter activity

regulation of gene expression; protein-DNA interactions

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