Transcriptional repression directed by the yeast α2 protein in vitro

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Abstract
The alpha 2 protein, a homeodomain protein involved in specifying cell type in the budding yeast Saccharomyces cerevisiae, is a transcriptional repressor. alpha 2 binds cooperatively with Mcm1, a serum response factor-related protein, to the a-specific gene operator. The alpha 2-Mcm1 complex in turn recruits Ssn6 and Tup1 to the operator, and we believe that these latter two proteins are responsible for the transcriptional repression. Placement of the a-specific gene operator in any of a variety of positions upstream of a test promoter leads to repression of that promoter in vivo. In this respect, the a-specific gene operator resembles a negatively acting enhancer. Here we describe the in vitro reconstitution of this example of negative control from a distance. We observe repression in vitro in the absence of exogenously added activator protein and on templates that lack binding sites for known activator proteins, and we infer that alpha 2-directed repression acts on the general transcription machinery.