In many viruses an empty """"""""prohead"""""""" is assembled and subsequently filled with DNA by the action of ATP dependent portal motor. DNA packaging occurs in many phages, herpesviruses, adenoviruses and poxvirues and it is therefore an important target for anti-viral drug development. In this application, we propose to use genetic, biochemical and biophysical approaches to expand and deepen our previous single-molecule studies of the packaging process by the portal motor of bacteriophage F29. This phage is an ideal system to investigate this process as a robust in-vitro packaging assay has been available. During the packaging process the DNA is compacted to near-crystalline density-overcoming energetic penalties due to electrostatics repulsion, DNA bending stiffness, and entropy. Because this motor is comprised of a pentameric ring of ASCE ATPases, its study will shed important light into the operation of other members of this family and of the larger superfamily of AAA+ ring ATPases, known to be responsible for a large number of cellular functions, from protein unfolding and degradation to chromosomal segregation in prokaryotes. We propose to characterize in great detail the various chemical and mechanical events during the operational cycle of each ATPase (i.e., ATP binding, hydrolysis, product release, translocation, etc) and to establish the precise timing or coordination among the cycles of the individual subunits in the ring. We will also establish the ability of the motor to generate torque, its magnitude and its generation mechanism relative to the production of linear force. These studies will be complemented by a characterization of the nature and strength of the contacts made between the DNA and the motor and its modulation during the various phases of the mechanochemical cycle. Finally, we will establish the participation of other non-catalytic elements of the motor such as the head-tail connector and the regulation of the motor's dynamics by the internal DNA pressure generated inside the capsid during the packaging process. To carry out these studies we will take advantage of state-of-the-art optical tweezers instrumentation in our laboratory. This instrument will make it possible for us to follow the packaging process with the unprecedented spatial resolution of 1A with a temporal resolution of 1 sec. Results of biophysical measurements will be integrated with structure determination by x-ray crystallography and cryo-electron microscopy (from established collaborations) and used to guide the development of models of this process.
We propose to study the detailed molecular mechanisms of the packaging motor responsible for genome compaction in the bacteriophage 29 using genetic, biochemical and biophysical approaches. Specifically we will use a single molecule optical tweezers approach to characterize the coordination between the individual chemical cycles of the five subunits in this ring ATPase. We will also establish the ability of this motor to generate torques and the nature and strength of its interaction with the DNA. Finally, we wish to investigate how the increasing internal DNA pressure regulates the dynamics of the motor during packaging.
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