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.<br/><br/>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.<br/><br/>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.