Mechanism of Genome Packaging in Bacteriophage T4

该项目将确定病毒的DNA遗传物质被包装在衣壳内的机制。噬菌体T4是一种感染大肠杆菌的病毒，它将被用作分析DNA包装中涉及的分子马达的功能的模型。这项研究还将对理解在生物体中执行不同功能的分子马达具有广泛的意义。这些包括：DNA凝聚到染色体中，分子进出细胞，以及肌肉收缩中的机械运动。这项研究还可能开辟发现新型抗病毒药物的途径，并设计生物纳米马达将治疗分子输送到细胞中。这项研究将成为指导高中、本科生、研究生和博士后等多个教育层次学生的极佳模式。学生将接触到最新的遗传、生化和生物物理方法，并与在跨学科研究领域拥有专业知识的研究人员互动。学生们将在有关噬菌体和病毒组装的国际会议上展示他们的研究成果。大型DNA病毒，如尾巴噬菌体和疱疹病毒，利用强大的包装机器将DNA强行转移到衣壳中。在噬菌体T4a171kb，56微米长，DNA被包装在接近结晶的树状结构中，形成一个大小为120×86纳米的衣壳。包装机由三个关键部件组成：十二聚体入口gp20、五聚体马达gp17和寡聚体调节器gp16。马达装配在位于壳体特殊五重顶点的入口上。利用来自ATP水解的能量，该马达以每秒高达~2000个碱基对的速度引起DNA运动，并基于遗传和生化研究提出了一个详细的机制。在本项目中，将通过提出一些基本问题来深入探讨包装机的功能。如何将23？DNA一端插入40？门户频道？马达如何产生60 pNewton力，将高带电的DNA分子限制在一个“微小的”衣壳容器中？导致DNA机械运动的马达-DNA相互作用的动力学是什么？发动机和门户如何交互和通信？运动亚单位是否以协调的方式激活ATPase？运动域是如何在毫秒级同步的？涉及遗传学、生物化学、结构和单分子生物物理学的互补实验室的多学科方法将被整合起来回答这些问题。这些结果可能有助于在接近原子分辨率的情况下重建病毒DNA包装马达的详细机制。

This project will determine the mechanisms by which the DNA genetic material of a virus is packaged inside a capsid. Bacteriophage T4, a virus that infects Escherichia coli, will be used as a model to analyze the functions of a molecular motor involved in DNA packaging. This research will also have broad implications to the understanding of molecular motors that carry out diverse functions in living organisms. These include: condensation of DNA into chromosomes, transport of molecules into and out of cells, and mechanical motion in muscle contraction. The research might also open avenues to discover novel antivirals, and to engineer biological nanomotors to deliver therapeutic molecules into cells. The research will serve as an excellent model to mentor students at many levels of education; high school, undergraduate, graduate, and post-doctoral. Students will be exposed to the most current genetic, biochemical, and biophysical approaches, and interact with investigators having expertise in interdisciplinary research areas. The students will present their research findings in international conferences on phage and virus assembly.Large DNA viruses such as the tailed bacteriophages and herpes viruses employ powerful packaging machines to forcefully translocate DNA into the capsid. In bacteriophage T4 a 171-kb, 56 micrometer-long, DNA is packaged at near crystalline densite into a capsid of size 120 by 86 nanometers. The packaging machine consists of three key components: a dodecameric portal, gp20; a pentameric motor, gp17; and an oligomeric regulator, gp16. The motor is assembled on the portal, which is located at the special five-fold vertex of the capsid. Utilizing the energy derived from ATP hydrolysis, the motor causes DNA motion at a rate of up to ~2000 base pairs per second, and a detailed mechanism has been proposed based on genetic and biochemical studies. In the current project, the functions of the packaging machine will be probed deeply by asking some fundamental questions. How is a 23Å DNA end threaded into a 40Å portal channel? How does the motor generate 60 pNewtons force necessary to confine the highly charged DNA molecule in a "tiny" capsid container? What are the dynamics of motor-DNA interactions that lead to mechanical motion of DNA? How do the motor and portal interact and communicate? Do the motor subunits fire ATPases in a coordinated fashion? How are the motor domains synchronized at millisecond timescale? Multidisciplinary approaches from complementary laboratories involving genetics, biochemistry, structure, and single molecule biophysics will be integrated to answer these questions. The results may help reconstitute the detailed mechanism of a virus DNA packaging motor at near atomic resolution.