Combinatorial Interactions among Eukaryotic Transcription Factors

高等真核生物的启动子往往具有复杂的结构。在酿酒酵母中，糖酵解酶基因的上游激活序列（UAS）元件与高等真核生物的增强子和近端启动子非常相似，因为它们由几种不同转录因子的多个结合位点组成。每个蛋白质结合位点本身显示很少的UAS活性，但它们一起形成强大的UAS元件。据信在糖酵解基因的UAS元件上聚集的转录因子包括Abflp、Reb1p、Rap1p、Gcr1p、Gcr2p和Ga111p。这些蛋白的单独作用以及它们如何相互作用形成酵母中最强的启动子尚不清楚。本研究的目的是提供有关这些转录因子的精确作用的信息。具体而言：i) Abflp和Reb1p结合的功能是清除邻近结合位点的核小体，从而允许其他蛋白质进入其结合位点的假设得到验证。ii)研究了糖酵解基因UAS元件中Rap1p-和Gcr1p-结合位点之间空间关系的重要性。iii)利用gcr2和gal11突变的酵母菌株来评估辅助因子Gcr2p和Gal11p在从TATA-box中置换核小体中的作用。iv)研究可能影响转录因子结合糖酵解基因启动子能力的环境因素。这些实验将为单个激活蛋白如何在真核生物启动子中以组合方式相互作用以介导高水平表达提供重要的新信息。***基因表达的主要控制点之一是转录调控。一种被称为RNA聚合酶的酶负责将遗传信息从DNA转化为RNA。启动子是DNA中的一个区域，它需要将RNA聚合酶带到基因上，这样它们就可以被转录。它们由TATA-box、近端启动子和增强子或上游激活序列（UAS）组成。近端启动子和增强子或UAS元件是DNA中蛋白质结合的位点。在这些序列上结合的蛋白质通过影响TATA-box上发生的事件来激活转录。本研究的重点是阐明在酵母中糖酵解酶基因的UAS元件上组装的蛋白质所起的确切作用。在酵母中，编码糖酵解酶的基因表达量最高。酵母糖酵解基因的充分表达需要大量的多功能蛋白。虽然这些蛋白质在糖酵解基因的UAS元件之间的组合相互作用已经被很好地记录下来，但控制它们相互作用的机制尚未建立。本研究的目的是：i)检验序列结合假设。ii)确定空间关系的重要性。iii)调查被认为影响塔塔箱事件的辅助因素的作用。iv)研究可能影响转录因子结合糖酵解基因UAS元件从而调控表达能力的环境因素。% % %

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. %%%