SAXS STUDIES ON ENDOGENEOUS HUMAN TFID

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。在真核生物中，至少有80种蛋白质参与了转录前起始复合物（PIC）的建立，该复合物足以识别启动子并启动高水平的受调控转录。道尔顿大小的转录复合物和结构转换定义了调控基因表达的机制。后来，我们使用电子显微镜来确定人TFIID（14个亚基，2 MDa）复合物的低分辨率结构。目前的生化分析表明，激活剂诱导的构象变化，在我们的TFIID/TFIIA复合物。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实验将使我们能够研究这种活化剂/TFIID/TFIIA相互作用在溶液中的动态性质，最终目标是定义条件，以启动这些大分子组装物的结晶试验。将先前通过凝胶过滤、电子显微镜和动态光散射确定为均质的浓缩免疫纯化TFIID（1 mg/ml）在含有20 mM Hepes pH 7.9和300 mM谷氨酸钾的缓冲液中稀释至0.1 mg/ml。将在不同浓度的TFIID下收集散射曲线，并进行分析以确定TFIID复合物在这些条件下是否聚集。

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.