Mechanism of ATP-driven DNA Packaging in Bacteriophage T4

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。大型DNA病毒、细菌病毒（“噬菌体”或“噬菌体”）和真核病毒（如疱疹病毒）将病毒基因组包装在蛋白质外壳（衣壳）内。感染大肠杆菌的噬菌体T4将一个171-kb的56微米长的DNA包裹在一个120 x 86纳米的衣壳中。包装后的DNA高度有序，其密度（~ 500mg /ml）几乎与晶体DNA相当。一个强大的包装马达由两种非结构蛋白组成，小端酶gp16 （18 kDa）和大端酶gp17 (69 kDa)，利用ATP水解能将DNA易位到衣壳中。马达组装在二十面体衣壳的特殊顶点，称为入口顶点。最近的分子遗传学和生化分析导致：1)定义了DNA易位的功能基序；Ii)获得部分组分的原子结构；阐明电机通过松弛和紧张构象状态交替产生静电力的机理。在这个项目中，将确定包装电机各个部分的生化和机械化学特性——铰链、DNA凹槽、离子对和传感器。催化位点的动态变化将使用在催化过程的不同步骤中有缺陷的突变体来梳理。运动功能，如步长，滑动和暂停，以及亚单位之间的协调将深入分析。当机器封装DNA时，电机各部分的运动将被实时解析。多种方法，包括分子遗传学，生物化学，结构和单分子生物物理学将被用来产生一个接近原子水平的包装机制的理解。更广泛的影响：对噬菌体T4 DNA包装机制的理解解决了病毒生命周期中的一个核心问题。该研究将有助于理解生物体将ATP化学能转化为机械运动的机制，以及细胞分裂过程中DNA的浓缩和解浓缩。它可能为发现新的抗病毒药物和设计马达将分子输送到细胞中开辟道路。噬菌体T4系统将作为一个优秀的实验模型，用于指导高中、本科、研究生和博士后等多个教育层次的学生。学生将接触到各种前沿的生物学和生物物理学方法，并与具有跨学科问题专业知识的研究人员互动。学生们将在噬菌体和病毒组装的国际会议上展示他们的研究成果。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Large DNA viruses, bacterial viruses ("bacteriophages" or "phages") and eukaryotic viruses such as herpes viruses, package the viral genome inside a protein shell (capsid). The bacteriophage T4, which infects E. coli, packages a 171-kb 56 micrometer long DNA into a 120 x 86 nanometer capsid. The packaged DNA is highly ordered and its density (~500 mg/ml) nearly equals that of crystalline DNA. A powerful packaging motor consisting of two non-structural proteins, the small terminase gp16 (18 kDa) and the large terminase gp17 (69 kDa), translocates DNA into the capsid utilizing ATP hydrolysis energy. The motor is assembled at the special vertex of the icosahedral capsid known as the portal vertex. Recent molecular genetics and biochemical analyses led to: i) defining the functional motifs involved in DNA translocation; ii) obtaining the atomic structures of some of the components; and iii) elucidating mechanism in which the motor generates electrostatic force by alternating between relaxed and tensed conformational states. In this project, the biochemical and mechanochemical properties of various parts of the packaging motor -- the hinge, the DNA grooves, the ion pairs, and the sensors -- will be determined. The dynamic changes at the catalytic site will be teased out using mutants that are defective at different steps of the catalytic process. Motor functions such as step size, slipping and pausing, and coordination among subunits will be analyzed in depth. The movements of various parts of the motor as the machine packages DNA will be resolved in real time. Multiple approaches, including molecular genetics, biochemistry, structure, and single molecule biophysics will be employed to generate a near atomic level understanding of the packaging mechanism.Broader impacts: Understanding of the phage T4 DNA packaging mechanism addresses one of the central questions in the life cycle of a virus. The research will contribute to the understanding of the mechanisms by which living organisms convert ATP chemical energy into mechanical motion, and condense and de-condense DNA during cell division. It might open avenues to discover novel antivirals and to engineer the motor to deliver molecules into cells. The phage T4 system will be used as an excellent experimental model to mentor students at many levels of education: high school, undergraduate, graduate and post-doctoral. Students will be exposed to a variety of cutting edge biological and biophysical approaches, and interact with investigators having expertise in interdisciplinary problems. The students will present their research in international conferences on phage and virus assembly.