Combinatorial Interactions Among Eukaryotic Transcriptional Factors

BakerCombinatorial转录因子之间的相互作用是一个共同的主题，但在大多数情况下，这些相互作用的机制是未知的。在酿酒酵母的糖酵解酶基因上游激活序列（UAS）元件中，阻遏物激活蛋白1（Rap 1 p）和糖酵解调节蛋白1（Gcr 1 p）一起形成一些已知的最强的启动子结合复合物。将阐明Rap 1 p和Gcr 1 p之间的组合相互作用的性质和机制。Rap 1 p作为多功能转录因子的模型，这些转录因子能够在转录中发挥看似相反的作用，根据其结合位点的序列背景作为激活剂或抑制剂发挥作用。Gcr 1 p作为一类序列特异性转录因子的模型，在体外对其结合位点显示高亲和力但低特异性。关于Rap 1 p和Gcr 1 p，有迹象表明它们都可能参与与其他结合伴侣的组合相互作用。在糖酵解酶基因UAS元件中，Rap 1 p促进Gcr 1 p的结合。为了确定Rap 1 p促进Gcr 1 p结合的机制，将测试结合协同性的替代模型。蛋白质-蛋白质相互作用模型和DNA弯曲模型将被研究。一个基因组的方法将被用来确定其他蛋白质参与GCR 1 P的组合相互作用。酵母基因组阵列将用于识别细胞内依赖于Gcr 1 p完全表达的每个基因。Gcr 1 p依赖性基因的调控区域将被搜索以寻找共同的序列基序。将研究与Gcr 1 p结合位点相邻的常见序列基序，以检验它们是Gcr 1 p结合伴侣的结合位点的假设。将进行体内足迹法和定点诱变，以确认推定结合位点的重要性。一旦序列基序被确定为结合位点，它们将被用于鉴定来自噬菌体pegda-gt 11酵母表达文库的克隆，所述噬菌体pegda-gt 11酵母表达文库表达与序列基序结合的蛋白。鉴定编码DNA结合活性的噬菌体克隆将直接导致鉴定编码Gcr 1 p的其他结合伴侣的酵母基因。这些实验将定义Rap 1 p和Gcr 1 p之间发生的DNA结合相互作用的确切性质，并为Gcr 1 p的DNA结合结构域的结构研究奠定基础。转录调控是基因表达的一个重要控制点，是将DNA中的信息复制到mRNA中的过程。该项目的目标是确定两种蛋白质之间的相互作用，这些蛋白质调节酵母基因的转录，这些基因编码分解葡萄糖的蛋白质。酿酒酵母已被证明是研究基因表达不可或缺的模型系统。这些实验将确定这两种蛋白质相互作用以及与DNA相互作用以调节基因表达的分子机制。此外，还将鉴定可能受类似机制调控的其他基因。这项工作将为阐明细胞内转录调控蛋白之间的相互作用网络奠定基础。

BakerCombinatorial interactions between transcription factors are a common theme, yet in most cases the mechanisms governing these interactions are not known. At glycolytic enzyme gene upstream activating sequence (UAS) elements of Saccharomyces cerevisiae, repressor activator protein 1 (Rap1p) and glycolysis regulatory protein 1 (Gcr1p) come together to form some of the strongest promoter-binding complexes known. The properties and mechanisms responsible for combinatorial interactions between Rap1p and Gcr1p will be elucidated. Rap1p serves as a model for multi-functional transcriptional factors that are capable of carrying out seemingly opposite roles in transcription, functioning as both an activator or repressor depending on the sequence context of their binding sites. Gcr1p serves as a model for a class of sequence-specific transcription factors that display high affinity but low specificity for their binding sites in vitro. With regard to Rap1p and Gcr1p, there are indications that they both may participate in combinatorial interactions with other binding partners as well. At glycolytic enzyme gene UAS elements, Rap1p facilitates the binding of Gcr1p. To determine the mechanism by which Rap1p facilitates Gcr1p's binding, alternate models for binding cooperativity will be tested. A protein-protein interaction model and a DNA-bending model will be investigated. A genomic approach will be used to identify other proteins that participate in combinatorial interactions with Gcr1p. Yeast genomic arrays will be used to identify every gene within the cell that is dependent on Gcr1p for full expression. The regulatory regions of Gcr1p-dependent genes will be searched for common sequence motifs. Common sequence motifs adjacent to Gcr1p-binding sites will be investigated to test the hypothesis that they are the binding sites for binding partners of Gcr1p. Both in vivo footprinting and site-directed mutagensis will be undertaken to confirm the importance of the putative binding sites. Once the sequence motifs are established as binding sites, they will be used to identify clones from a phage lambda-gt11 yeast expression library expressing proteins that bind to the sequence motif. Identification of the phage clones encoding the DNA-binding activity will lead directly to the identification of yeast genes encoding other binding partners of Gcr1p. These experiments will define the precise nature of the DNA binding interactions that occur between Rap1p and Gcr1p and will lay the ground work for structural studies on the DNA-binding domain of Gcr1p. This work will also lay a foundation for elucidating the network of combinatorial interactions within cells.A major control point of gene expression is the regulation of transcription, the process by which the information in DNA is copied into mRNA. The goal of this project is to define the interactions between two proteins that regulate the transcription of yeast genes that encode proteins that break down glucose. The yeast Saccharomyces cerevisiae has proved indispensable as a model system for studies of gene expression. The experiments will determine the molecular mechanisms by which these two proteins interact with each other and with DNA to regulate gene expression. Furthermore, additional genes that may be regulated by similar mechanisms will be identified. This work will lay a foundation for elucidating the network of interactions between transcriptional regulatory proteins that occur within cells.