BIOCHEMICAL ANALYSIS OF FACTORS ASSOCIATED WITH TFIID

与 TFIID 相关因素的生化分析

• 批准号: 2415261
• 负责人: NAOKO TANESE
• 金额: $ 24.76万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415261
• 关键词: HeLa cells; affinity chromatography; chimeric proteins; complementary DNA; gene induction /repression; genetic library; immunoprecipitation; intermolecular interaction; molecular cloning; protein biosynthesis; protein reconstitution; protein structure function; recombinant proteins; recombinant virus; transcription factor; transfection; transfection /expression vector; vaccinia virus

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.

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 和基础成分结合的因子 转录机器。 定义位点特异性转录的精确机制 激活转录的因素一直是人们关注的焦点 调查。 有人提出转录激活剂可能 靶向预引发复合物组装过程中的多个步骤。 TFIID 就是这样的一个靶标，它是一种多亚基复合物，由 TATA盒结合蛋白（TBP）和多个紧密相关的 因子（TAF），作为介导转录的共激活因子 激活。 拟议研究的总体目标是 识别并描述 TAF 和 TAF 之间的功能关系 激活剂。 长期目标是重建转录 重组 TFIID 亚基和激活剂使分子事件 导致转录激活的定义可以更精确。 第一个具体目标涉及功能特性的检查 最近克隆的人类TAF130。 由于 TAF 的功能是 共激活剂，预计它们直接接触激活剂。 初步研究表明 hTAF130 与人类结合 体外转录因子 Sp1。 hTAF130 和 Sp1 的结构域 结合所需的亲和层析将被表征 酵母的方法和遗传筛选。 其他激活剂将被测试 用于与 TAF130 的结合及其相互作用的表征。 这 这些 TAF-激活剂相互作用的重要性将在 转录的上下文。 为此，部分 TFIID 复合体 用纯化的重组 TAF 蛋白和 TBP 进行体外重构 将在激活剂存在的情况下在转录测定中进行测试 与 TAF130 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。 为了 出于分析目的，TFIID 的亚基将瞬时表达为 使用感染/转染方案和复合物的 HeLa 细胞 将通过脉冲标记和免疫沉淀来监测组装。 这种方法的一个潜在优势是效率高且易于使用 可以分析突变蛋白的功能。 对于大 大规模生产重组蛋白，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 TAF95 蛋白使用酵母中的 GAL4 两种杂交系统。 人类 cDNA 表达库将用于筛选以识别潜力 hTAF95 的相互作用靶点。 hTAF95 包含多个副本 WD40 重复序列，一个与蛋白质-蛋白质相互作用有关的基序。 将在体外测试候选克隆与 hTAF95 的相互作用 它们的功能在转录测定中进行了检查。 拟议的研究 可能有助于揭示转录的一般机制 激活。 这些发现与定义分子 正常细胞生长、分化和发育过程中发生的事件 发展以及癌症和其他人类疾病。