DNA-bound Bas1 recruits Pho2 to promoters to stimulate expression of the ADE genes in Saccharomyces cerevisiae.
Indrani Som, Jennifer Urbanowski, Ronda Rolfes
Department of Biology, Georgetown University, 37th and O Street NW, Washington, DC, 20057-1229, USA
Expression of the ADE regulon of Saccharomyces cerevisiae is repressed by the presence of exogenous purines. Derepression requires the transcription activators Bas1 and Pho2 as well as the pathway intermediate SAICAR. Intracellular adenine nucleotide pools indirectly regulate the expression of the ADE regulon by feedback inhibiting flux through the pathway, leading to low levels of SAICAR. However, when feedback inhibition is relieved, increased flux through the pathway increases the levels of SAICAR. SAICAR promotes interaction between the transcription factors by an unknown mechanism. In this study, we investigated if nuclear localization and binding to promoter DNA are regulated by purines. Using indirect immunofluorescence, we found that Bas1 is localized to the nucleus under both repressing and derepressing conditions. Importantly, Bas1 is bound to promoter DNA under both repressing and derepressing conditions as assessed by chromatin immunoprecipitation (ChIP) assays. When we measured the binding of Bas1 to wild type and mutant promoters in vitro and in vivo, we found that Bas1 binds independently to each of its two sites with equal affinity. Pho2 was not required for DNA association of Bas1, but it was required for both transcription and an increase in Bas1-associated DNA by ChIP. Although Bas1 is localized to promoters independent of adenine, Pho2 is recruited to promoters only under derepressing conditions when the ADE genes are transcribed.
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