Mechanism of Genome Packaging in Bacteriophage T4

Catholic University Of America, Washington DC

Investigators

Abstract

This project will determine the mechanisms by which the DNA genetic material of a virus is packaged inside a capsid. Bacteriophage T4, a virus that infects Escherichia coli, will be used as a model to analyze the functions of a molecular motor involved in DNA packaging. This research will also have broad implications to the understanding of molecular motors that carry out diverse functions in living organisms. These include: condensation of DNA into chromosomes, transport of molecules into and out of cells, and mechanical motion in muscle contraction. The research might also open avenues to discover novel antivirals, and to engineer biological nanomotors to deliver therapeutic molecules into cells. The research will serve as an excellent model to mentor students at many levels of education; high school, undergraduate, graduate, and post-doctoral. Students will be exposed to the most current genetic, biochemical, and biophysical approaches, and interact with investigators having expertise in interdisciplinary research areas. The students will present their research findings in international conferences on phage and virus assembly. 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 at near crystalline densite into a capsid of size 120 by 86 nanometers. The packaging machine consists of three key components: a dodecameric portal, gp20; a pentameric motor, gp17; and an oligomeric regulator, gp16. The motor is assembled on the portal, which is located at the special five-fold vertex of the capsid. Utilizing the energy derived from ATP hydrolysis, the motor causes DNA motion at a rate of up to ~2000 base pairs per second, and a detailed mechanism has been proposed based on genetic and biochemical studies. In the current project, the functions of the packaging machine will be probed deeply by asking some fundamental questions. How is a 23Å DNA end threaded into a 40Å portal channel? How does the motor generate >60 pNewtons force necessary to confine the highly charged DNA molecule in a "tiny" capsid container? What are the dynamics of motor-DNA interactions that lead to mechanical motion of DNA? How do the motor and portal interact and communicate? Do the motor subunits fire ATPases in a coordinated fashion? How are the motor domains synchronized at millisecond timescale? Multidisciplinary approaches from complementary laboratories involving genetics, biochemistry, structure, and single molecule biophysics will be integrated to answer these questions. The results may help reconstitute the detailed mechanism of a virus DNA packaging motor at near atomic resolution.