SAXS STUDIES ON ENDOGENEOUS HUMAN TFID

关于人类内源性 TFID 的 SAXS 研究

• 批准号: 7370518
• 负责人: ROBERT S COLEMAN
• 金额: $ 0.24万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7370518
• 关键词: SAXS; STUDIES; ENDOGENEOUS; HUMAN; TFID

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In eukaryotes, at least 80 proteins have been implicated in establishing a transcription pre-initiation complex (PIC) sufficient to recognize a promoter and initiate high levels of regulated transcription Structural studies combined with functional biochemistry of purified and reconstituted transcription complexes will be essential to fully understand the dynamic assembly of these mega-dalton sized transcription complexes and the structural transitions defining the mechanisms regulating gene expression. reviously we had used electron microscopy to determine the low resolution structure of a human TFIID(14 subunits, 2MDa) complex. Current biochemical assays indicate an activator induced conformational change in our TFIID/TFIIA complex. We propose to initiate SAXS studies to confirm and extend our EM studies on TFIID. Unlike our EM work, SAXS experiments will allow us to examine the dynamic nature of this activator/TFIID/TFIIA interaction in solution with the eventual goal of defining conditions to initiate crystallization trials on these large macromolecular assemblies. Concentrated immunopurified TFIID(1mg/ml) that was previously determined to be homogenous by gel filtration, electron microscopy and dynamic light scattering will be diluted down to 0.1mg/ml in a buffer containing 20mM Hepes pH 7.9 and 300mM potassium glutamate. Scattering curves will be collected at various concentrations of TFIID and analyzed to determine if the TFIID complex is aggregated under these conditions.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。在真核生物中，至少有 80 种蛋白质参与建立转录前起始复合物 (PIC)，足以识别启动子并启动高水平的受调控转录。结构研究与纯化和重构转录复合物的功能生物化学相结合，对于充分了解这些兆道尔顿大小的转录复合物的动态组装以及定义调节基因表达机制的结构转变至关重要。之前我们使用电子显微镜来确定人类 TFIID（14 个亚基，2MDa）复合物的低分辨率结构。目前的生化分析表明，激活剂诱导了我们的 TFIID/TFIIA 复合物的构象变化。我们建议启动 SAXS 研究来确认和扩展我们对 TFIID 的 EM 研究。与我们的 EM 工作不同，SAXS 实验将使我们能够检查溶液中激活剂/TFIID/TFIIA 相互作用的动态性质，最终目标是定义在这些大分子组装体上启动结晶试验的条件。先前通过凝胶过滤、电子显微镜和动态光散射确定为均质的浓缩免疫纯化 TFIID (1mg/ml) 将在含有 20mM Hepes pH 7.9 和 300mM 谷氨酸钾的缓冲液中稀释至 0.1mg/ml。将收集不同浓度的 TFIID 的散射曲线并进行分析以确定 TFIID 复合物在这些条件下是否聚集。