REGULATION OF BACTERIOPHAGE P2 LATE GENE EXPRESSION

本申请中提出的研究旨在了解 温带噬菌体晚期基因表达机制的研究 P2.P2晚期启动子与正常序列不太相似 被大肠杆菌RNA聚合酶识别，尽管宿主酶是 后期转录所必需的。P2晚期的正常表达 基因需要P2DNA复制基因和 P2 OGR基因。P4激活P2晚期基因需要 P4增量基因的产物，并在 与正常P2晚期基因表达相同的部位。功能界别 P2晚期基因启动子的结构域将通过缺失来定义 分析和体外诱变。两国之间的相互作用 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 额外的P2编码因子，似乎需要 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。基因中的一个点突变 大肠杆菌RNA聚合酶的α亚基特异性阻断P2 晚期基因转录。额外的RNA聚合酶突变体 映射到阿尔法亚单位并影响P2后期转录将是 与世隔绝的，有特点的。氨基酸取代的影响 OGR中的蛋白质将使用不同的琥珀进行研究 抑制者。温带噬菌体P2和P2的相互作用 卫星噬菌体P4提供了一个独特的模型系统来研究 对大肠杆菌RNA聚合酶启动子特异性的调控，以及 应该会导致对RNA聚合酶如何识别 DNA中的序列。

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.