BIOCHEMICAL ANALYSIS OF FACTORS ASSOCIATED WITH TFIID

描述（改编自申请人的摘要）：转录 激活涉及序列特异性转录的相互作用 与启动子DNA结合的因子和基底膜的组分 转录机器 确定位点特异性转录的精确机制 因子激活转录一直是激烈的主题 调查 已经提出转录激活因子可以 在前起始复合体的组装过程中瞄准多个步骤。 一个这样的目标似乎是TFIID，一种多亚基复合物， TATA盒结合蛋白（TBP）和多个紧密相关的 因子（TAF），其作为介导转录的共激活因子， activation. 拟议研究的总体目标是 确定和描述TAF之间的功能关系， 活化剂。 长期目标是重建转录， 重组TFIID亚基和激活剂， 导致转录激活的定义可以更精确。 第一个具体目标涉及功能特性的检查 最新克隆的人类TAF 130 由于TAF的功能是 在共活化剂中，预测它们直接接触活化剂。 初步研究表明，hTAF 130与人 体外转录因子Sp1。 hTAF 130和Sp1的结构域 将通过亲和色谱法表征结合所需的量。 方法和酵母中的遗传筛选。 将测试其他激活剂 用于结合TAF 130并表征它们的相互作用。 的 这些TAF-激活剂相互作用的重要性将在 转录的背景。 为此，部分TFIID复合物 用纯化的重组TAF蛋白和TBP体外重构 将在存在激活剂的情况下在转录测定中进行测试 与TAF 130结合 第二个具体目标是设计一种使用牛痘的体内系统， 用于有效组装TFIID复合物的病毒载体。 为 出于分析目的，TFIID的亚基将在细胞中瞬时表达。 使用感染/转染方案和复合物的HeLa细胞 通过脉冲标记和免疫沉淀监测组装。 这种方法的一个潜在优势是效率和易用性， 可以分析这些突变蛋白的功能。 大型 重组蛋白的规模生产，HeLa细胞将 与表达TFIID各亚基的重组病毒共感染 部分复合物将被纯化用于进一步的生化 分析包括体外转录和DNA结合测定。 第三个具体目标是确定克隆人的目标 TAF 95蛋白在酵母中使用GAL 4双杂交系统。 人cDNA 表达文库将用于筛选以鉴定潜在的 hTAF 95的相互作用靶标。 hTAF 95含有几个拷贝的 WD 40重复序列，一种与蛋白质相互作用有关的基序。 将测试候选克隆与hTAF 95的体外相互作用， 它们的功能在转录测定中检测。 拟议研究 可能会揭示一些转录的一般机制， activation. 这些发现与定义分子 在正常细胞生长、分化和分化期间发生的事件， 发展，以及癌症和其他人类疾病。

DESCRIPTION (Adapted from Applicant's Abstract): Transcriptional activation involves an interplay of sequence- specific transcription factors bound to the promoter DNA and the components of the basal transcription machinery. Defining the precise mechanism by which site-specific transcription factors activate transcription has been the subject of intense investigation. It has been proposed that transcriptional activators may target multiple steps during the assembly of a preinitiation complex. One such target appears to be TFIID, a multi-subunit complex consisting of the TATA box-binding protein (TBP) and multiple tightly associated factors (TAFs), which serve as coactivators for mediating transcriptional activation. The general objective of the proposed research is to identify and characterize the functional relationship between TAFs and activators. The long-term goal is to reconstitute transcription with recombinant TFIID subunits and activators so that the molecular events leading to transcriptional activation may be defined more precisely. The first specific aim involves examination of the functional properties of the recently cloned human TAF130. Since TAFs function as coactivators, it is predicted that they directly contact the activators. Preliminary studies suggest that hTAF130 binds to the human transcription factor Sp1 in vitro. The domains of hTAF130 and Sp1 required for binding will be characterized by affinity chromatographic methods and genetic screens in yeast. Other activators will be tested for binding to TAF130 and their interactions characterized. The significance of these TAF-activator interactions will be examined in the context of transcription. Towards this end, a partial TFIID complex reconstituted in vitro with purified recombinant TAF proteins and TBP will be tested in transcription assays in the presence of the activators that bind to TAF130. The second specific aim is to devise a system in vivo using vaccinia viral vectors for efficient assembly of the TFIID complex. For analytical purposes, subunits of TFIID will be transiently expressed in HeLa cells using the infection/transfection protocol and complex assembly will be monitored by pulse-labeling and immunoprecipitation. A potential advantage of this approach is the efficiency and ease with which the mutant proteins may be analyzed for function. For the large scale production of the recombinant proteins, HeLa cells will be coinfected with the recombinant viruses expressing each subunit of TFIID and the partial complexes will be purified for further biochemical analyses including in vitro transcription and DNA binding assays. The third specific aim is to identify the target of the cloned human TAF95 protein using the GAL4 two hybrid system in yeast. A human cDNA expression library will be used in the screen to identify the potential interacting target(s) of hTAF95. hTAF95 contains several copies of the WD40 repeat, a motif implicated in protein-protein interactions. Candidate clones will be tested for interaction with hTAF95 in vitro and their function examined in transcription assays. The proposed research may shed some light into the general mechanism of transcriptional activation. These findings are relevant to defining the molecular events occurring during normal cell growth, differentiation and development, as well as in cancer and other human diseases.