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.
