PARALLEL PATHWAYS OF GENE REGULATION

基因调控的平行途径

• 批准号: 2186125
• 负责人: David J Stillman
• 金额: $ 15.57万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186125
• 关键词: DNA binding protein; DNA footprinting; Saccharomyces cerevisiae; binding proteins; cell cycle; chimeric proteins; fungal genetics; gene deletion mutation; gene expression; gene interaction; genetic models; genetic promoter element; genetic regulation; genetic transcription; laboratory rabbit; model design /development; molecular cloning; nucleic acid sequence; protein structure; transcription factor

The goal of this project is to determine the molecular mechanisms which isolate pathways of transcriptional regulation, using the unicellular eukaryote Saccharomyces cerevisiae. We have identified two pathways of transcriptional regulation which show many parallels. The HO and CTS1 genes show identical patterns of cell cycle regulation, with expression in late G1 phase of the cell cycle. These genes are regulated by zinc-finger containing transcription factors, SWI5 and ACE2. These two transcription factors show identical patterns of cell cycle regulated transcription and cell cycle regulated nuclear localization. Furthermore, the DNA binding domains of SWI5 and ACE2 are identical, and they both bind to the HO promoter in vitro. However, despite these many similarities, SWI5 and ACE2 regulate different genes in vivo. SWI5 activates transcription of HO but not of CTS1, and ACE2 activates CTS1 but not HO. We propose to investigate the mechanisms which isolate the SWI5/HO and ACE2/CTS1 pathways. We have data supporting two distinct models for regulation, and these two models are not mutually exclusive. The first model proposes that different negative regulators bind to the HO and CTS1 promoters, and that SWI5 and ACE2 differ in their ability to overcome the repressive effects of these promoter specific negative regulators. In vivo analysis of promoter constructs provides support for this model. The second model proposes that additional factors are required for specificity in promoter recognition, and that cooperative interactions between factors contributes to promoter binding and pathway isolation. In support of this model we have identified a factor which binds cooperatively, along with SWI5, to the HO promoter. We postulate the existence of an analogous factor which is needed for specific promoter recognition of the CTS1 promoter by ACE2. Experiments are proposed to analyze the interactions of these factors with sites on DNA and with each other, and to test models of regulatory control. The factor that binds DNA cooperatively with SWI5 has been purified. The gene encoding this factor has been cloned, and we know this factor is a homeodomain protein. Cooperative interactions have not been observed before between a zinc finger protein and a homeodomain protein, and we propose to characterize their interaction. Experiments are proposed to identify regions of these proteins required for cooperative interactions.

该项目的目标是确定分子机制， 转录调节分离途径， 使用单细胞 真核生物酿酒酵母 我们已经确定了两种途径， 转录调控有许多相似之处。 HO和CTS 1 基因显示相同的细胞周期调控模式，表达 处于细胞周期的G1晚期。 这些基因受 含锌指转录因子SWI 5和ACE 2。 这两 转录因子显示相同的细胞周期调控模式， 转录和细胞周期调控的核定位。 此外，SWI 5和ACE 2的DNA结合结构域是相同的， 它们都在体外与HO启动子结合。 然而，尽管有许多 SWI 5和ACE 2在体内调节不同的基因。 SWI5 激活HO的转录，但不激活CTS 1的转录，并且ACE 2激活CTS 1 而不是HO。 我们建议研究分离SWI 5/HO的机制， ACE 2/CTS 1通路。 我们有数据支持两种不同的模型， 这两种模式并不相互排斥。 第一 模型提出，不同的负调节剂结合HO和CTS 1 启动子，SWI 5和ACE 2在克服这些缺陷的能力上不同。 这些启动子特异性负调控因子的抑制作用。 在 启动子构建体的体内分析为该模型提供了支持。的 第二个模型提出，需要额外的因素， 启动子识别特异性和协同相互作用 因子之间的相互作用有助于启动子结合和途径分离。 为了支持这个模型，我们已经确定了一个因素， 沿着SWI 5，与HO启动子协同作用。 我们假设 存在特异性启动子所需的类似因子 通过ACE 2识别CTS 1启动子。 实验被提议为 分析这些因素与DNA上位点的相互作用， 其他，并测试监管控制模型。 与SWI 5协同结合DNA的因子已被纯化。 的 编码这个因子的基因已经被克隆出来，我们知道这个因子是一个 同源域蛋白 尚未观察到合作互动 锌指蛋白和同源结构域蛋白之间的联系， 建议描述他们的互动。 实验被提议为 确定这些蛋白质的合作相互作用所需的区域。