REGULATION OF BACTERIOPHAGE P2 LATE GENE EXPRESSION

噬菌体 P2 晚期基因表达的调控

• 批准号: 3286002
• 负责人: GAIL E CHRISTIE
• 金额: $ 16.37万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-05 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3286002
• 关键词: DNA directed RNA polymerase; Escherichia coli; affinity chromatography; coliphages; gene expression; genetic promoter element; genetic transcription; molecular cloning; mutant; nucleic acid sequence; plasmids; prophages; protein structure; tissue /cell culture; transfection; virus envelope; virus genetics; virus protein

The studies proposed in this application are aimed at understanding the mechanisms for late gene expression in the temperate coliphage P2. P2 late promoters do not resemble sequences normally recognized by E. coli RNA polymerase, although the host enzyme is required for late transcription. Normal expression of P2 late genes requires the P2 DNA replication genes and the product of the P2 ogr gene. Transactivation of P2 late genes by P4 requires the product of the P4 delta gene and initiates transcription at the same sites as normal P2 late gene expression. The functional domains of P2 late gene promoters will be defined by deletion analysis and in vitro mutagenesis. The interactions between purified P2 ogr protein, late promoters and host RNA polymerase will be investigated using chemical and kinetic methods. An additional P2-encoded factor which appears to be required for expression of P2 late genes will be isolated and its effect on late gene transcription will be studied in vitro. A point mutation in the alpha subunit of E. coli RNA polymerase specifically blocks P2 late gene transcription. Additional RNA polymerase mutants which map to the alpha subunit and affect P2 late transcription will be isolated and characterized. The effects of amino acid substitution in the ogr protein will be studied using different amber suppressors. The interactions between temperate coliphage P2 and satellite phage P4 provide a unique model system for studying the modulation of promoter specificity of E. coli RNA polymerase, and should lead to new insights into how RNA polymerase recognizes sequences in DNA.

本申请中提出的研究旨在了解 温带大肠杆菌噬菌体晚期基因表达的机制 P2。 P2 晚期启动子与正常序列不相似 被大肠杆菌 RNA 聚合酶识别，尽管宿主酶是 后期转录所需。 P2晚期正常表达 基因需要P2 DNA复制基因及其产物 P2食人魔基因。 P4 反式激活 P2 晚期基因需要 P4 delta 基因的产物并在 与正常 P2 晚期基因表达相同的位点。 功能性的 P2晚期基因启动子的结构域将通过删除来定义 分析和体外诱变。 之间的相互作用 纯化的 P2 ogr 蛋白、晚期启动子和宿主 RNA 聚合酶 将使用化学和动力学方法进行研究。 一个 似乎需要额外的 P2 编码因子 将分离 P2 晚期基因的表达及其对晚期的影响 基因转录将在体外进行研究。 点突变 大肠杆菌 RNA 聚合酶的 α 亚基特异性阻断 P2 晚期基因转录。 其他 RNA 聚合酶突变体 映射到 α 亚基并影响 P2 晚期转录 分离并表征。 氨基酸取代的影响 在ogr蛋白中将使用不同的琥珀进行研究 抑制器。 温带大肠杆菌噬菌体 P2 和 卫星噬菌体 P4 为研究噬菌体提供了独特的模型系统 调节大肠杆菌RNA聚合酶的启动子特异性，以及 应该会对 RNA 聚合酶如何识别产生新的见解 DNA 中的序列。