BIOCHEMICAL ANALYSIS OF FACTORS ASSOCIATED WITH TFIID

描述(改编自申请者摘要)：抄本 激活涉及序列特异性转录的相互作用 与启动子DNA结合的因子和基础成分 转录机器。 确定位点特异性转录的精确机制 激活转录的因子一直是激烈研究的主题 调查。已经有人提出，转录激活剂可以 靶向预引发复合体组装过程中的多个步骤。 其中一个目标似乎是TFIID，它是一个由多个亚基组成的复合体 TATA盒结合蛋白(TBP)和多个紧密相关的 因子(TAFs)，作为调节转录的辅助激活因子 激活。拟议研究的总体目标是 识别和表征TAFs和TAFs之间的功能关系 激活剂。长期目标是通过以下方式重建转录 重组TFIID亚基和激活剂使分子事件 导致转录激活的定义可能更准确。 第一个具体目标涉及功能属性的检查 最近克隆的人类TAF130。由于TAFs的功能是 共激活剂，据预测，他们直接接触激活剂。 初步研究表明hTAF130与人 体外转录因子Sp1。HTAF130和Sp1的结构域 所需的结合将通过亲和层析进行表征 酵母菌的方法和遗传筛选。其他激活剂将接受测试 用于与TAF130结合并表征其相互作用。这个 这些TAF-激活剂相互作用的意义将在 转录的上下文。为此，一个部分的TFIID复合体 纯化的重组TAF蛋白与TBP的体外重组 将在有激活剂存在的情况下进行转录检测 它与TAF130绑定在一起。 第二个具体目标是设计一种体内使用牛痘病毒的系统。 高效组装TFIID复合体的病毒载体。为 出于分析目的，TFIID的亚基将在 应用感染/转染法和复合体的HeLa细胞 组装将通过脉冲标记和免疫沉淀进行监测。 这种方法的一个潜在优势是效率和易用性 突变的蛋白质可能会被分析其功能。对于大型企业而言 重组蛋白的规模化生产，HeLa细胞将 与表达TFIID各亚基的重组病毒共感染 部分络合物将被提纯以进行进一步的生化 分析包括体外转录和DNA结合分析。 第三个具体目标是识别克隆人的目标 利用GAL4双杂交系统在酵母中表达TAF95蛋白。一个人的基因 将在屏幕上使用表达文库来识别潜在的 HTAF95的相互作用靶点(S)。HTAF95包含多个副本 WD40重复，一个与蛋白质相互作用有关的基序。 候选克隆将在体外测试与hTAF95的相互作用，并 它们的功能通过转录检测进行了检测。拟议的研究 可能会对转录的一般机制有所帮助。 激活。这些发现与定义分子 发生在正常细胞生长、分化和 在人类发展、癌症和其他人类疾病方面也是如此。

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.