I will present our recent results on the packaging of DNA by the connector motor at the base of the head of bacteriophage f29. As part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid to insure its proper transport and delivery to other host cells. Bacteriophage f29 packages its 6.6 mm long double-stranded DNA into a 42 nm dia. x 54 nm high capsid via a portal complex that possesses 5 ATPases that hydrolyze ATP. This process is remarkable because entropic, electrostatic, and bending energies of the DNA must be overcome to package the DNA to near-crystalline density. We have used optical tweezers to pull on single DNA molecules as they are packaged, thus demonstrating that the portal complex is a force generating motor. We find that this motor can work against loads of up to ~57 picoNewtons on average, making it one of the strongest molecular motors ever reported. Movements of over 5 mm are observed, indicating high processivity. Pauses and slips also occur, particularly at higher forces. We establish the force-velocity relationship of the motor and find that the rate-limiting step of the motor's cycle is force dependent even at low loads. Interestingly, the packaging rate decreases as the prohead is filled, indicating that an internal pressure builds up due to DNA compression. We estimate that at the end of the packaging the capsid pressure is ~6 MegaPascals, corresponding to an internal force of ~52 pN acting on the motor. The biological implications of this internal pressure and the mechano-chemical efficiency of the engine are discussed. We have also investigated the coordination between the mechanical and the chemical steps in the operation of the motor and have been able to propose the first putative cycle for this molecular machine. We determine, within this cycle, the step at which the chemical energy is converted into mechanical work and we have characterized the nature of the interactions between the motor and the DNA. Finally, high resolution optical tweezers experiments are also making it possible for us to investigate in detail the operation of this motor and the coordination among the ATPases during the overall motor cycle.
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