Grabbing the cat by the tail: Studies of DNA packaging by single phi29 bacteriophage particles using optical tweezers

PHYS 186

Carlos Bustamante, Quantitative Biology Institute/Department of Physics, University of California, Berkeley/Howard Hughes Medical Inst, 608A Stanley Hall #3220, Berkeley, CA 94720-3220
As part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid to insure proper transport and delivery to other host cells. Bacteriophage_phi29 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. 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 packaging the capsid pressure is ~6 MegaPascals, corresponding to an internal force of ~52 pN acting on the motor. We have also investigated the coordination between the mechanical and the chemical steps in the motor operation and have proposed 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 characterize the nature of the interactions between the motor and the DNA. Finally, high resolution optical tweezers experiments are enabling us to investigate in detail the operation of this motor and the coordination among ATPases during the overall cycle.