Combinatorial Interactions among Eukaryotic Transcription Factors

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Abstract 9404721 Promoters in higher eukaryotes tend to have complex structures. In Saccharomyces cerevisiae the upstream activating sequence (UAS) elements of glycolytic enzyme genes closely resemble enhancers and proximal promoter elements of higher eukaryotes in that they are composed of multiple binding sites for several different transcription factors. Each protein binding site by itself displays little UAS activity, but together they form powerful UAS elements. The transcription factors believed to assemble at the UAS elements of glycolytic genes include Abflp, Reb1p, Rap1p, Gcr1p, Gcr2p, and Ga111p. The individual roles of the proteins and how they interact to form the strongest promoters in yeast are not known. The goal of this study is to provide information as to the precise roles played by several of these transcription factors. Specifically: i) The hypothesis that binding of Abflp and Reb1p functions to clear nucleosomes from adjacent binding sites, thereby allowing other proteins access to their binding sites is tested. ii) The importance of spatial relationships between Rap1p- and Gcr1p- binding sites in the UAS elements of glycolytic genes is investigated. iii) Yeast strains with mutations in gcr2 and gal11 are utilized to assess the roles of the co-factors Gcr2p and Gal11p in displacing nucleosomes from the TATA-box. iv) Environmental factors which may influence the ability of transcription factors to bind at glycolytic gene promoters are investigated. These experiments will provide important new information as to how the individual activator proteins interact in a combinatorial fashion at eukaryotic promoters to mediate high-level expression. *** One of the major control points of gene expression is the regulation of transcription. An enzyme known as RNA polymerase is responsible for converting the genetic information from DNA to RNA. Promoters are regions in the DNA which are required to bring RNA polymerase to genes so they can be t ranscribed. They are made of sequences known as the TATA-box, proximal promoters, and enhancers or upstream activating sequences (UAS). Proximal promoters and enhancers or UAS elements are sites in the DNA to which proteins bind. Proteins that bind at these sequences activate transcription by influencing events that occur at the TATA-box. The focus of this study is to elucidate the precise roles played by the proteins that assemble at the UAS elements of glycolytic enzyme genes in the yeast, Saccharomyces cervisiae. In yeast, genes encoding glycolytic enzymes are the most highly expressed. A number of abundant, multifunctional proteins are required for full expression of yeast glycolytic genes. While combinatorial interactions between these proteins at the UAS elements of glycolytic genes has been well documented, the mechanism(s) governing their interactions have yet to be established. The aims of this study are: i) To test a sequential binding hypothesis. ii) To determine the importance of the spatial relationships. iii) To investigate the role of cofactors which are believed to influence events at the TATA-box. iv) To investigate environmental factors which may influence the ability of transcription factors to bind at glycolytic gene UAS elements, thus regulating expression. %%%