Combinatorial Interactions Among Eukaryotic Transcriptional Factors

转录因子之间的组合相互作用是一个共同的主题，但在大多数情况下，控制这些相互作用的机制尚不清楚。在酿酒酵母糖酵解酶基因上游激活序列(UAS)元件上，抑制子激活蛋白1(Rap1p)和糖酵解调节蛋白1(Gcr1p)共同形成了一些已知最强的启动子结合复合体。将阐明Rap1p和Gcr1p之间的组合相互作用的性质和机制。Rap1p是多功能转录因子的模型，这些转录因子在转录过程中扮演着看似相反的角色，根据其结合位点的序列背景，既可以作为激活因子，也可以作为抑制因子发挥作用。Gcr1p是一类序列特异性转录因子的模型，这些转录因子在体外对其结合部位表现出高亲和力但低特异性。关于Rap1p和Gcr1p，有迹象表明它们也可能参与与其他结合伙伴的组合相互作用。在糖酵解酶基因UAS元件中，Rap1p促进了Gcr1p的结合。为了确定Rap1p促进Gcr1p结合的机制，将测试结合协同性的替代模型。将研究蛋白质-蛋白质相互作用模型和DNA弯曲模型。基因组学方法将被用来识别参与与Gcr1p组合作用的其他蛋白质。酵母基因组阵列将被用来识别细胞内依赖Gcr1p充分表达的每一个基因。将搜索依赖于Gcr1p的基因的调节区，以寻找共同的序列基序。我们将研究Gcr1p结合位点附近的常见序列基序，以检验它们是Gcr1p结合伙伴的结合位点的假设。体内足迹和定点突变都将被用来确认推测的结合位点的重要性。一旦序列基序被确定为结合位点，它们将被用于从表达与序列基序结合的蛋白质的噬菌体lambda-gt11酵母表达文库中鉴定克隆。对编码DNA结合活性的噬菌体克隆的鉴定将直接导致对编码Gcr1p其他结合伙伴的酵母基因的鉴定。这些实验将确定Rap1p和Gcr1p之间发生的DNA结合相互作用的确切性质，并将为Gcr1p的DNA结合域的结构研究奠定基础。这项工作还将为阐明细胞内的组合相互作用网络奠定基础。基因表达的一个主要控制点是转录调控，即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，这两种蛋白质调节酵母基因的转录，而酵母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缺少的模型系统。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，还将确定可能受类似机制调控的其他基因。这项工作将为阐明发生在细胞内的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.