REGULATION OF BACTERIOPHAGE P2 LATE GENE EXPRESSION

本申请中提出的研究旨在了解 温性大肠杆菌噬菌体中晚期基因表达的机制 P2. P2晚期启动子与正常序列不相似 E.大肠杆菌RNA聚合酶，虽然宿主酶是 需要后期转录。 P2晚期正常表达 基因需要P2 DNA复制基因和P2 DNA复制基因的产物。 P2 ogr基因。 P4对P2晚期基因的反式激活需要： P4 δ基因的产物，并在转录起始位点启动转录。 与正常P2晚期基因表达相同的位点。 功能 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。 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，这似乎是必要的 P2晚期基因的表达将被分离，其对晚期 将在体外研究基因转录。 中的点突变 E.大肠杆菌RNA聚合酶特异性阻断P2 晚期基因转录 另外的RNA聚合酶突变体， 映射到α亚基并影响P2后期转录， 分离并表征。 氨基酸替代的影响 将使用不同的琥珀酰亚胺 抑制剂 温性大肠杆菌噬菌体P2与 卫星噬菌体P4提供了一个独特的模型系统， 调节E. coli RNA聚合酶，和 这将使我们对RNA聚合酶如何识别 DNA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.