PHAGE T4 HEAD ASSEMBLY AND INITIATION OF INFECTION

噬菌体 T4 头部组装和感染起始

• 批准号: 2059805
• 负责人: LINDSAY W BLACK
• 金额: $ 27.03万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-07-01 至 1997-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2059805
• 关键词: DNA virus; affinity labeling; bacterial genetics; bacteriophage T4; capsid; conformation; crosslink; enzyme mechanism; fusion gene; host organism interaction; membrane proteins; microorganism growth; microorganism metabolism; molecular pathology; mutant; protein biosynthesis; protein sequence; virus DNA; virus envelope; virus genetics; virus infection mechanism

The research focuses on the mechanism of viral DNA packaging and on the construction of phage T4 in vitro packaging-derived cloning vectors. Our work is especially directed toward 1) understanding the structures and functions of the multifunctional phage T4 packaging (prohead and terminase) proteins. The 3- dimensional basis for enzymatic (DNA translocating) and structural (prohead form-determining) roles of the major capsid protein gp23*), of a minor processed enzymatic product (gp23**), and of capsid gene mutants will be determined. The assembly of the DNA entrance (also the prohead initiation) vertex as an integral membrane protein will be examined using overexpression vectors; the role of a catalytic gene product and host systems in the membrane insertion will be determined. Specific packaging mutations in both these prohead proteins will be selected following gene-directed mutagenesis. The DNA terminase proteins which interact with the DNA entrance vertex protein in the prohead have been overexpressed and purified-their mechanisms of action in DNA translocation and concatemer cutting will be investigated. lambda and lambda-pBR322 derivative DNAs can be packaged into T4 heads in vitro. cos and/or P1 DNAs packaged into T4 and recircularized with the homologous site-specific recombination systems should allow development of T4-hybrid megacosmid vectors. We have proposed a novel "spiral-fold" model for packaged phage DNA. Morphological and (with phage ) chemical work can definitively establish this model. Packaged kinked or non-B form DNA will be detected, and its interaction with the DNA binding site in the major capsid protein and its minor processed enzymatic product (gp23**) will be probed. We will determine what DNA structures can be packaged in vivo and in vitro; e.g. whether nicks and heteroduplex loops are excluded from packaged DNA in vitro, and whether the terminase proteins act to discriminate against such structures in coupled DNA repair processes.

研究的重点是病毒DNA包装的机制 并构建T4噬菌体体外包装衍生体 克隆载体。我们的工作特别针对1) 了解多功能的结构和功能 噬菌体T4包装(前部和终止酶)蛋白。3- 酶的空间基础(DNA转位)和 主要衣壳的结构(前部形状决定)作用 蛋白质gp23*)，次要加工酶产品(gp23**)， 衣壳基因突变体的数量将会确定。组装的 DNA入口(也称为前部启动)顶点作为 整体膜蛋白将通过过度表达进行检测 载体；催化基因产物和宿主系统在 膜的插入将被确定。特定包装 这两个前导蛋白的突变都将被选择 在基因导向的突变之后。DNA终止酶 与DNA进入顶点蛋白相互作用的蛋白质在 前言被过度表达和提纯-他们的 DNA易位和串联反应的作用机制 将对切割进行调查。Lambda和Lambda-pBR322 在体外，衍生的DNA可以被包装成T4头部。科司 和/或包装成T4的P1DNA，并与 同源位点特定重组系统应该允许 T4-杂交型巨链载体的研制。我们已经提议 一种新的包装噬菌体DNA的螺旋折叠模型。 形态和(利用噬菌体)化学作用可以确定 建立这一模式。包装的扭结或非B形式的DNA将是 以及它与DNA结合部位的相互作用 主要衣壳蛋白及其次要加工酶产品 (gp23**)将被探测。我们将确定什么DNA结构 可在体内和体外包装；例如，NICKS和 在体外包装的DNA中排除了异源双链环，并且 终止酶蛋白是否起到了歧视这种 耦合DNA修复过程中的结构。