MECHANISMS OF TRANSCRIPTIONAL REGULATION IN EUKARYOTES

真核生物转录调控机制

• 批准号: 2838556
• 负责人: James T. Kadonaga
• 金额: $ 25.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2838556
• 关键词: DNA directed RNA polymerase; Drosophilidae; HeLa cells; developmental genetics; embryology; enhancer binding protein; eukaryote; gel mobility shift assay; gene expression; genetic enhancer element; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; laboratory rabbit; nucleic acid sequence; protein purification; transcription factor

DESCRIPTION: The long-term objective of this project is to understand the mechanism of basal transcription by RNA polymerase II. This grant specifically proposes to use both biochemical and genetic approaches to study the function of a conserved, downstream basal promoter element termed the DPE (downstream promoter element). The DPE is a distinct 7 bp element that is located at about +30 (typically, from +25 to +34) relative to the transcription start site. It is present in many TATA-less promoters and is specifically bound by TFIID, but not by the TATA-binding protein (TBP). The available data suggest that the DPE is a conserved core promoter element that is, in many respects, a downstream counterpart to the TATA box. The specific aims are as follows. 1. Biochemical characterization of the DPE. These studies involved the analysis of the characteristics of functionally active DPE elements as well as the nature of the physical interaction of TFIID with TATA-less, DPE-containing promoters. 2. Purification and characterization of a DPE-specific basal transcription activity. Basal transcription of TATA-less, DPE-containing promoters requires an activity that is distinct from the factors that are sufficient for transcription of TATA-containing promoters (i.e., this activity is distinct from TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II). This aim is focused on the purification and characterization of this activity, which is tentatively named DSF (DPE-specific factor). 3. Analysis of the DPE in vivo in transgenic Drosophila. Enhancer trapping techniques will be used to gain insight into the biological role of the DPE. In particular, these experiments will test the ability of a broad range of enhancers to regulate transcription differentially from TATA-containing, DPE-less (TATA+ Inr+ DPE-) promoters relative to TAT-less, DPE-containing (TATA- Inr+ DPE+) promoters. The experiments proposed here would contribute to our fundamental understanding of gene expression and would be applicable to the study of human diseases, such as AIDS and many forms of cancer, that might be treated by the specific and directed control of transcription.

描述：该项目的长期目标是了解 RNA聚合酶II的基础转录机理。 这笔赠款 专门提议使用生化和遗传学方法 研究称为保守的下游启动子元素的功能 DPE（下游启动子元素）。 DPE是一个独特的7 bp元素 相对于 转录起始站点。 它存在于许多无塔塔的发起人中，是 特别由TFIID约束，但不受TATA结合蛋白（TBP）的结合。 这 可用数据表明DPE是保守的核心启动子元素 也就是说，在许多方面，是塔塔框的下游对应物。 这 具体目标如下。 1。DPE的生化表征。 这些研究涉及对功能特征的分析 主动DPE元素以及物理相互作用的性质 带有无TATA的含DPE启动子的TFIID。 2。纯化和 DPE特异性基础转录活性的表征。 基础 无TATA的转录，含DPE的启动子需要活动 这与足以转录的因素不同 含Tata的启动子（即，此活动与TFIIA不同， tfiib，tfiID，tfiie，tfiif，tfiih和RNA聚合酶II）。 这个目标是 专注于此活动的纯化和表征， 暂定命名为DSF（DPE特异性因素）。 3。对DPE的分析 转基因果蝇中的体内。 增强器捕获技术将用于 深入了解DPE的生物学作用。 特别是这些 实验将测试各种增强器调节的能力 转录与含TATA的无DPE差异（tata+ inr+） DPE-）启动子相对于含DPE的含DPE（tata-inr+ dpe+） 发起人。 这里提出的实验将有助于我们 对基因表达的基本理解，将适用于 研究人类疾病，例如艾滋病和多种形式的癌症，可能 可以通过特定和定向的转录控制来处理。