Mechanism of ATP-driven DNA Packaging in Bacteriophage T4

噬菌体和疱疹病毒的双链DNA包装是一个令人着迷的生物学问题。在T4噬菌体中，一个56毫米的DNA被转移到一个预组装的衣壳中，并被组织成一个高度有序的凝聚体，其包装密度相当于一个DNA晶体。gp16 （18 kDa）和gp17 （69 kDa）两种非结构端酶/包装蛋白识别并切割T4序列DNA，并通过与位于独特顶点的十二聚体门脉蛋白（gp20, 61 kDa）相互作用将其连接到前体。因此，一个DNA包装机被创造出来，它利用ATP水解能将DNA易位到衣壳中。在一个头部长度的基因组被包装后，末端酶进行第二次切割，与dna全头部分离，与新产生的末端结合，并将其连接到第二个前导，以一种持续的方式继续包装。据报道，噬菌体DNA包装马达是迄今为止测量到的最强的发力生物马达之一。是哪个部件驱动了这台非凡的机器？先前的研究表明，大端酶蛋白gp17表现出较弱的atp酶活性，这种活性被小端酶蛋白gp16刺激了50倍。分子遗传学、生物化学和计算方法已经在gp17中定义了一个n端atp酶催化中心，并将其刺激的atp酶活性与DNA易位联系起来。在这个项目中，使用一个综合的包装模型，将解决基本问题：i) gp16和gp17中的哪些结构域和氨基酸残基相互作用引起atp酶刺激？ii)包装机中端酶蛋白的化学计量是什么？iii) atp酶催化途径中的哪个转变与DNA易位相关？iv) atp酶亚基是否通过顺序机制“激发”？v)旋转偶联是否是DNA易位的潜在机制？一组独特的三磷酸腺苷酶突变体将被用来进行定义和定量的生物化学，并梳理出包装机制的分子细节。终端酶和包装马达的低温电镜和x射线结构分析，以及单DNA分子包装研究，以测试旋转包装模型，将补充生化研究。因此，该项目旨在解决噬菌体和病毒DNA包装的核心机制问题。它将促进对病毒如何在宿主细胞中包装遗传物质的理解，这是成功将基因组传递到新宿主的基本要求。更广泛的影响：这个项目的结果将对DNA包装领域和atp酶马达产生广泛的影响。此外，这个包含分子遗传学、生物化学、生物物理、计算和结构方法的多维项目将成为培养本科生和研究生的一个令人兴奋的模型，并为未来的研究事业奠定坚实的基础。学生将有一个独特的机会独立设计实验，批判性地分析一个明确的问题，并应用丰富的T4噬菌体遗传学和组装信息来解决它。该项目将为学生提供令人兴奋的机会，在国际会议上展示他们的研究成果，如噬菌体和病毒大会，以及美国微生物学会。与普渡大学、东京工业大学和日本冈崎国立研究所的研究人员合作，将拓宽学生的视野，并促进与这些机构的学生研究人员和资深科学家的互动。本项目中使用的概念将在分子生物学实验室教授给大一本科生，这是所有生物学专业和许多学科的医学预科学生的要求。该项目的研究生将担任该实验室的助教，这有助于他们提高教学技能，并将他们在研究实验室中学到的知识传授给一群崭露头角的本科科学家。

Double stranded DNA packaging in bacteriophages and herpes viruses is a fascinating biological problem. In phage T4, a 56 mm DNA is translocated into a pre-assembled capsid and organized into a highly ordered condensate having a packing density equivalent to that of a DNA crystal. Two non-structural terminase/packaging proteins, gp16 (18 kDa) and gp17 (69 kDa), recognize and cut T4 concatemeric DNA and link it to the prohead by interacting with the dodecameric portal protein (gp20, 61 kDa) situated at the unique vertex. A DNA packaging machine is thus created, which translocates DNA into the capsid utilizing ATP hydrolysis energy. After one headful length genome is packaged, the terminase makes a second cut, dissociates from the DNA-full head and associates with the newly generated end and links it to a second prohead to continue packaging in a processive manner. Phage DNA packaging motor was reported to be one of the strongest force-generating biological motors measured to date. Which component powers this extraordinary machine? Previous studies showed that the large terminase protein gp17 exhibits a weak ATPase activity, which is stimulated by 50-fold by the small terminase protein gp16. Molecular genetic, biochemical, and computational approaches have defined an N-terminal ATPase catalytic center in gp17 and linked its stimulated ATPase activity to DNA translocation. In this project, using a comprehensive packaging model, fundamental questions will be addressed: i) Which domains and amino acid residues in gp16 and gp17 interact to cause ATPase stimulation? ii) What is the stoichiometry of terminase proteins in the packaging machine? iii) Which transition in the ATPase catalytic pathway is coupled to DNA translocation? iv) Are the ATPase subunits "fired" by a sequential mechanism? v) Is rotational coupling the underlying mechanism of DNA translocation? A unique collection of well-characterized ATPase mutants will be used to perform defined and quantitative biochemistry and tease out the molecular details of the packaging mechanism. Collaborative cryo-EM and X-ray structural analyses of terminase and packaging motor, and single DNA molecule packaging studies to test the rotational packaging models, will complement the biochemical studies. Thus, the project is designed to address core mechanistic questions of phage and viral DNA packaging. It will advance the understanding of how viruses package genetic material in the host cell, a fundamental requirement for successfully transmitting the genome into a new host.Broader Impact: Results from this project will have broad implications to the DNA packaging field and ATPase motors in general. Additionallly, this multi-dimensional project, encompassing molecular genetic, biochemical, biophysical, computational, and structural approaches would serve as an exciting model to train undergraduate and graduate students and establish a solid foundation for future research careers. The students will have a unique opportunity to design experiments independently, critically analyze a defined question, and apply the rich T4 phage genetics and assembly information to solve it. The project will provide exciting opportunities for the students to present their research in International conferences such as Phage and Virus Assembly, and American Society for Microbiology. Collaborations with researchers at the Purdue University, Tokyo Institute of Technology, and Okazaki National Research Institute, Japan, will broaden students' horizons and foster interactions with fellow student researchers and senior scientists from these institutions. Concepts used in this project will be taught to freshman undergraduate students in the molecular biology laboratory, which is a requirement for all biology concentrators and pre-med students from many disciplines. Graduate students from this project will serve as Teaching Assistants for this lab, which helps them to develop teaching skills and allows transfer of what they learned in the research lab to a group of budding scientists at the undergraduate level.