Mechanism of Genome Packaging in Bacteriophage T4

该项目将确定包含遗传物质的病毒DNA如何被包装在称为衣壳的病毒隔室中，以促进病毒的传播和感染。噬菌体T4是一种感染大肠杆菌的病毒，将被用作分析负责DNA包装的分子机器的模型。这项研究将对理解强大的分子马达具有广泛的意义，这些分子马达在生物体中执行许多不同的功能，如基因组的凝聚、分子进出细胞的运输以及肌肉收缩的机械运动。该研究还将成为指导高中、本科、研究生和博士后等不同教育水平学生的典范。学生将参与最前沿的遗传、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领先研究人员进行互动。该项目将为学生提供机会，在噬菌体和病毒组装的国际会议上展示他们的研究，并共同促进那些可能没有这种接触的人参与STEM领域。大型DNA病毒，如尾状噬菌体和疱疹病毒，使用强大的包装机器将DNA强行转移到衣壳中。在T4噬菌体中，一个171kb， 56微米长的DNA被包装在120 x 86纳米的衣壳中，接近晶体密度。包装机由三个关键部件组成：五聚体电机gp17 (69 kDa)；十二轴通道gp20 (61 kDa)；寡聚调节因子gp16 （18 kDa）。马达组装在入口上，入口位于病毒衣壳的特殊五重顶点。马达利用ATP水解产生的能量，以高达~ 2000bp /秒的速度以活塞式运动将DNA易位到衣壳中。在当前的项目中，将通过提出一些基本问题来分析DNA包装的机制。马达是如何抓住DNA的？它是如何产生力的？DNA每一步移动多少埃？运动亚基合作移动DNA吗？哪些运动区域移动，移动了多远？传送门的确切功能是什么？门户是否像阀门一样，与马达一起抓住和释放DNA ？来自互补实验室的多学科方法将采用复杂的分子遗传学，生物化学和单分子生物物理学方法来回答这些问题。该结果可能导致病毒DNA包装马达在近原子分辨率上的详细机制。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project will determine how virus DNA, which contains the genetic material, is packaged inside a viral compartment called the capsid to facilitate viral propagation and infection. Bacteriophage T4, a virus that infects the bacterium Escherichia coli, will be used as a model to analyze a molecular machine that is responsible for DNA packaging. This research will have broad implications for understanding powerful molecular motors that carry out numerous and diverse functions in living organisms such as condensation of genomes, transport of molecules into and out of cells, and mechanical motion in muscle contraction. The research will also serve as a model to mentor students at different levels of education: high school, undergraduate, graduate, and post-doctoral. Students will be involved in cutting-edge genetic, biochemical, and biophysical approaches, and interact with leading investigators having expertise in interdisciplinary research areas. The project will provide opportunities for students to present their research at international conferences on phage and virus assembly, and taken together, will promote participation in STEM fields for those who might not otherwise have this exposure.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 into a 120 x 86 nm capsid to near crystalline density. The packaging machine consists of three key components: a pentameric motor, gp17 (69 kDa); a dodecameric portal, gp20 (61 kDa); and an oligomeric regulator, gp16 (18 kDa). The motor is assembled on the portal, which is located at the special five-fold vertex of the virus capsid. Utilizing the energy derived from ATP hydrolysis, the motor translocates DNA into the capsid in a piston-like motion at a rate of up to ~2000 bp/sec. In the current project, the mechanism of DNA packaging will be analyzed by asking some fundamental questions. How does the motor grip the DNA? How does it generate force? How many Angstrom distance does the DNA move in each step? Do the motor subunits cooperate to move DNA? Which motor domains move and by what distance? What is the precise function of the portal? Does the portal act like a valve by gripping and releasing DNA in concert with the motor? Multidisciplinary approaches from complementary laboratories will employ sophisticated molecular genetics, biochemistry, and single molecule biophysics approaches to answer these questions. The results may lead to the detailed mechanism of a virus DNA packaging motor at near atomic resolution.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.