SAXS STUDIES ON ENDOGENEOUS HUMAN TFID

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在真核生物中，至少有80个蛋白质参与了转录预起始复合体(PIC)的建立，该复合体足以识别启动子并启动高水平的调控转录结构研究，结合纯化和重组的转录复合体的功能生物化学，对于全面理解这些百万道尔顿大小的转录复合体的动态组装和定义基因表达调控机制的结构转变是必不可少的。以前，我们用电子显微镜确定了人TFIID(14个亚基，2MDA)复合体的低分辨结构。目前的生化分析表明，激活剂诱导了我们的TFIID/TFIIA复合体的构象变化。我们建议启动SAXS研究，以确认和扩展我们对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这种激活剂/TFIID/TFIIA在溶液中相互作用的动态性质，最终目标是确定启动这些大分子组件结晶试验的条件。在含有20 mM HEPES pH 7.9和300 mM谷氨酸钾的缓冲液中，先前经凝胶过滤、电子显微镜和动态光散射确定为均一的浓缩免疫纯化的TFIID(1 mg/ml)将稀释到0.1 mg/ml。将收集不同浓度的TFIID的散射曲线并进行分析，以确定TFIID复合体在这些条件下是否聚集。

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.