Phage T4 Head Assembly and Initiation of Infection

噬菌体 T4 头部组装和感染启动

• 批准号: 6681705
• 负责人: LINDSAY W BLACK
• 金额: $ 37.13万
• 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 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6681705
• 关键词: DNA binding protein; DNA replication; Escherichia coli; bacteriophage T4; capsid; electron microscopy; genetic transcription; green fluorescent proteins; host organism interaction; nuclear magnetic resonance spectroscopy; protein biosynthesis; protein protein interaction; virus DNA; virus assembly; virus genetics; virus infection mechanism; virus protein

DESCRIPTION (provided by applicant): This grant application proposes continuation of research toward two main objectives: 1) understanding the mechanism of terminase catalyzed viral DNA translocation into the procapsid, and 2) determining the host resistance mechanism leading to the evolution of the CTS- (capsid targeting sequence) initiated set of polymorphic internal proteins injected by the T-even phages. The objectives will be furthered by development of a new phage-derived technique for assessing protein interactions. As part of the first objective, the DNA packaging mechanism will be investigated by focusing on terminase and portal dodecamer structure and function. A multimeric activated ATPase form of the terminase large subunit can be produced by incubation with the terminase small subunit and with antibody against denatured terminase large subunit. Together with portal or functional portaI-GFP (green fluorescent protein) fusion, the reconstituted packasome motor (terminase + portal + DNA) will be characterized. Work incorporating portaI-GFP and HOC-portal fusions into functional proheads and phage suggests that the portal dodecamer of the prohead can be immobilized without blocking DNA packaging. Continuation of this work can determine whether a favored portal rotation model for DNA packaging is valid. Preliminary work reveals that NMR can solve the structure of the small terminase subunit. T4 display has successfully identified a new terminase interactant, T4 late ( factor gp55, required for replication-coupled T4 late transcription. Recent work has established a dependence of DNA packaging on this component; we believe the interaction serves to load the large terminase subunit onto DNA. This interaction could also serve to tightly couple packaging to DNA repair at the late replication-dependent transcription stage of T4 development. Reconstitution of active in vitro T4 DNA packaging will employ isolated gene 55 defective in vivo concatemers as well as T4 transcription assay plasmids. These DNAs will be activated for packaging by loading late transcription-replication components. In the second objective, a diverse family of CTS-injected proteins has evolved by gene expansion at the IPI locus of the T-even phages to challenge a corresponding set of resistance genes in natural hosts of these viruses. We intend to determine the host resistance mechanism by analysis of two resistance gene(s), IBEGs and IBEGd, cloned from one pathogenic strain of E. coli. These two cloned genes are necessary and sufficient for phage T4 IPI- and other T-even phage resistance, and the two his-tag modified genes also confer resistance, thus facilitating study of the mechanism at the protein level.

描述（由申请人提供）：本授权申请提出继续研究两个主要目标：1）了解末端酶催化病毒DNA易位进入原衣壳的机制，以及2）确定导致CTS（衣壳靶向序列）进化的宿主抗性机制，该机制启动了由T-偶联物注入的一组多态性内部蛋白。通过开发一种新的噬菌体衍生技术来评估蛋白质相互作用，将进一步实现这一目标。作为第一个目标的一部分，DNA包装机制将集中在末端酶和门户十二聚体的结构和功能进行调查。可以通过与末端酶小亚基和针对变性末端酶大亚基的抗体孵育来产生末端酶大亚基的多聚体活化ATP酶形式。与门户或功能性门户-GFP（绿色荧光蛋白）融合物一起，将表征重构的包装体马达（末端酶+门户+ DNA）。将portaI-GFP和HOC-门户融合到功能性prohead和噬菌体中的工作表明，prohead的门户十二聚体可以被固定而不阻断DNA包装。这项工作的继续可以确定是否有利的门户网站旋转模型的DNA包装是有效的。初步工作表明，NMR可以解决的小末端酶亚基的结构。T4展示技术已经成功地鉴定出一种新的终止酶相互作用物，T4晚期（因子gp 55），它是复制偶联T4晚期转录所必需的。最近的工作已经建立了依赖于此组件的DNA包装，我们相信相互作用的大末端酶亚基加载到DNA上。这种相互作用也可以用于紧密耦合包装DNA修复在T4发育的后期复制依赖性转录阶段。活性体外T4 DNA包装的重建将使用分离的基因55缺陷体内多联体以及T4转录测定质粒。这些DNA将通过加载晚转录-复制组分而被激活用于包装。在第二个目标，一个不同的家庭的CTS注射蛋白质已经演变的基因扩增的IPI位点的T-甚至是挑战相应的一组抗性基因在这些病毒的天然宿主。本研究通过对从一株大肠杆菌中克隆的两个抗性基因IBEGs和IBEGd的分析，探讨了宿主的抗性机制。杆菌这两个克隆的基因是噬菌体T4 IPI-和其他T-甚至噬菌体抗性所必需的和足够的，并且两个His-标签修饰的基因也赋予抗性，从而促进在蛋白质水平上的机制的研究。