Combinatorial Interactions Among Eukaryotic Transcriptional Factors

真核转录因子之间的组合相互作用

• 批准号: 9816990
• 负责人: Henry Baker
• 金额: $ 37.5万
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9816990&HistoricalAwards=false
• 关键词: Combinatorial; Interactions; Among; Eukaryotic; Transcriptional

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.

转录因子之间的BakerCombinatorial相互作用是一个共同的主题，但是在大多数情况下，管理这些相互作用的机制尚不清楚。在糖酵解酶基因在糖含量酿酒酵母，抑制剂激活剂蛋白1（RAP1P）和糖酵解调节蛋白1（GCR1P）的元素中，上游激活序列（UAS）元素一起出现，形成了一些最有力的启动子结合体。将阐明负责RAP1P和GCR1P之间组合相互作用的特性和机制。 RAP1P是能够在转录中执行相反作用的多功能转录因子的模型，它取决于其结合位点的序列上下文，作为激活因子或阻遏物。 GCR1P是一类序列特异性转录因子的模型，这些模型在体外显示出高亲和力但对其结合位点的特异性较低。关于RAP1P和GCR1P，有迹象表明他们都可以参与与其他结合伙伴的组合相互作用。在糖酵解酶基因UAS元素上，RAP1P促进了GCR1P的结合。为了确定RAP1P促进GCR1P结合的机制，将测试用于结合合作的替代模型。将研究蛋白质 - 蛋白质相互作用模型和DNA弯曲模型。一种基因组方法将用于鉴定参与与GCR1P组合相互作用的其他蛋白质。酵母基因组阵列将用于识别依赖于GCR1P的细胞中的每个基因以进行全表达。 GCR1P依赖性基因的调节区域将被搜索以获取常见序列基序。将研究与GCR1P结合位点相邻的常见序列基序，以检验以下假设：它们是GCR1P结合伴侣的结合位点。体内足迹和定向诱变都将进行确认推定结合位点的重要性。一旦将序列基序确定为结合位点，它们将用于识别来自噬菌体lambda-gt11酵母表达式库，表达结合序列基序的蛋白质。编码DNA结合活性的噬菌体克隆的鉴定将直接导致鉴定编码GCR1P其他结合伴侣的酵母基因。这些实验将定义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结合结构域的结构研究奠定基础工作。这项工作还将为阐明细胞内组合相互作用网络的基础。基因表达的主要控制点是转录的调节，即DNA中的信息复制到mRNA中的过程。该项目的目的是定义两个蛋白质之间的相互作用，这些蛋白质调节了编码分解葡萄糖的蛋白质的转录。酿酒酵母的酵母菌已被证明是必不可少的基因表达研究模型系统。实验将确定这两种蛋白质相互作用并与DNA相互作用以调节基因表达的分子机制。此外，将确定可能通过相似机制调节的其他基因。这项工作将为阐明细胞内发生的转录调节蛋白之间的相互作用网络奠定基础。