Mechanism of ATP-driven DNA Packaging in Bacteriophage T4

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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.