BIOSYNTHESIS OF ADENOVIRUS EARLY RNAS

腺病毒早期 RNA 的生物合成

• 批准号: 2390630
• 负责人: ARNOLD J BERK
• 金额: $ 42.96万
• 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/2390630
• 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.

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