BIOSYNTHESIS OF ADENOVIRUS EARLY RNAS

腺病毒早期 RNA 的生物合成

• 批准号: 2087343
• 负责人: ARNOLD J BERK
• 金额: $ 42.48万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2087343
• 关键词: Adenoviridae; DNA binding protein; DNA directed RNA polymerase; HeLa cells; affinity chromatography; chemical binding; fluorescence polarization; gel filtration chromatography; gel mobility shift assay; genetic promoter element; genetic transcription; immunoprecipitation; laboratory rabbit; molecular cloning; mutant; posttranslational modifications; protein purification; site directed mutagenesis; transcription factor; virus DNA; virus genetics; virus protein; western blottings

The abnormal properties of cancer cells are due in part to the inappropriate activation of some transcription factors (TFs) and the inactivation of others. Understanding how TFs function should allow the design of therapies that modify the abnormal TFs that contribute to oncogenesis. Most regulatory TFs are modular proteins with distinct DNA binding and activation domains. The mechanisms by which DNA binding domains function are well understood, but little is known about how activation domains function. Activation domains stimulate pol Il initiation from a complicated preinitiation complex composed of general TFs and pol Il. We propose to study the activation domains of the strong viral activators adenovirus 2 E1A, Epstein-Barr Virus Zta, and herpes simplex virus VP16; as well as the important tumor suppressor p53. The studies depend on the ability to purify functional TFIID, the complex general transcription factor composed of the TATA-binding protein (TBP) and TAFs that initiates preinitiation complex assembly at promoters with a TATA-box. A minor nuclear protein, CR3BP, has been identified with the predicted properties of an E1A coactivator: it binds the wt E1A activation domain, but not to point mutants defective in activation that do bind TBP. If additional experimentation is consistent with E1A coactivator function, a cDNA encoding CR3BP will be cloned and used to analyze its transcriptional activity. Regions on the surface of TBP that interact with E1A and other activation domains, general TFs and TAFs will be analyzed by introducing amino acid substitutions into each of its 91 surface amino acid residues that do not contact DNA. TFIID containing mutant TBPs that bind general TFs and TAFs normally but are defective for activated transcription will be isolated and used in assays of activation domain binding and preinitiation complex assembly to determine which step in activated assembly, pol II initiation or promoter clearance is defective. Zta activates assembly of TFIID and TFIIA on promoter DNA, but this stimulation is not sufficient to account completely for Zta activation. Steps in preinitiation complex assembly subsequent to D-A assembly will be assayed using agarose gels capable of resolving DNA protein complexes of >10(6) Da and a gel filtration assay to detect factor binding to plasmid templates. Activation of pol II initiation and promoter clearance will also be analyzed. Similar studies will analyze activation by E1A and p53 and activation by combinations of activators on synthetic templates with binding sites for two types of activators. Specific antibodies raised against a recently cloned subunit of the pol III factor TFIIIC will be used to analyze the mechanism of TFIIIC regulation in response to viral infection and growth factors.

癌细胞的异常特性部分是由于 某些转录因子 (TF) 的不适当激活和 他人的失活。了解 TF 的功能应该允许 设计修改异常转录因子的疗法，这些转录因子有助于 肿瘤发生。大多数调控转录因子是具有不同 DNA 的模块化蛋白 结合域和激活域。 DNA 结合的机制 域的功能已被很好地理解，但对其如何实现却知之甚少 激活域功能。激活域刺激 pol II 由复杂的预引发复合体引发，该复合体由一般组成 TF 和 pol Il。我们建议研究激活域 强病毒激活剂腺病毒 2 E1A、Epstein-Barr 病毒 Zta 和 单纯疱疹病毒VP16；以及重要的肿瘤抑制因子 第 53 页。这些研究依赖于纯化功能性 TFIID 的能力， 由 TATA 结合组成的复杂通用转录因子 启动预起始复合物组装的蛋白质 (TBP) 和 TAF 在带有 TATA 盒的启动子处。一种次要核蛋白 CR3BP 确定了 E1A 共激活剂的预测特性： 结合 wt E1A 激活结构域，但不结合有缺陷的点突变体 在激活中确实结合TBP。如果额外的实验是 与 E1A 共激活子功能一致，编码 CR3BP 的 cDNA 将 被克隆并用于分析其转录活性。地区 与 E1A 和其他激活域相互作用的 TBP 表面， 一般的TF和TAF会通过引入氨基酸进行分析 对其 91 个表面氨基酸残基中的每一个进行替换 不接触DNA。 TFIID 含有结合一般 TF 的突变 TBP 和 TAF 正常但有激活转录缺陷 分离并用于激活结构域结合的测定和 预引发复合体组装以确定激活的哪一步 组装、pol II 启动或启动子清除有缺陷。兹塔 激活 TFIID 和 TFIIA 在启动子 DNA 上的组装，但这 刺激不足以完全解释 Zta 激活。 D-A 组装后的预启动复杂组装步骤将 使用能够解析 DNA 蛋白质的琼脂糖凝胶进行测定 >10(6) Da 的复合物和凝胶过滤测定法检测因子 与质粒模板结合。 pol II 启动的激活和 还将分析启动子清除率。类似的研究将分析 E1A 和 p53 激活以及激活剂组合激活 在具有两种类型激活剂结合位点的合成模板上。 针对最近克隆的 pol 亚基产生的特异性抗体 III因子TFIIIC将用于分析TFIIIC的作用机制 对病毒感染和生长因子的调节。