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Single Molecule Studies of Genome Packaging in Bacteriophage Phi29 Reveal Nonequilibrium DNA Dynamics and Continuous Allosteric Regulation of the Packaging Motor Complex


The high-density packaging of DNA in many viruses is a process that requires the action of powerful molecular motor complexes that couple the energy of ATP hydrolysis to the movement of DNA into the viral prohead. Besides being a key step in viral assembly, DNA packaging is a general model for understanding the physics of tightly confined polymers. A fundamental question raised in the literature is whether packaging can be modeled as a quasistatic thermodynamic process, in which the DNA relaxes quickly to equilibrium, or whether it involves nonequilibrium dynamics. The nature of the DNA dynamics at high prohead fillings and its relationship to the precise mechanochemical cycle of the packaging motor remain an open question. This work is aimed at investigating that relationship via single molecule optical tweezers experiments.

In Chapter II I report the results of experiments in which we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.

In Chapter III I report evidence for an unconventional type of allosteric regulation of a biomotor. We show that the genome-packaging motor of phage phi29 is regulated by a sensor that detects the density and conformation of the DNA packaged inside the viral prohead, and slows the motor by a mechanism distinct from the effect of a direct load force on the motor. Specifically, we show that motor-ATP interactions are regulated by a signal that is propagated allosterically from inside the viral shell to the motor mounted on the outside. This signal continuously regulates the motor speed and pausing in response to changes in either density or conformation of the packaged DNA, and slows the motor before the buildup of large forces resisting DNA confinement. Analysis of motor slipping reveals that the force resisting packaging remains low (<1 pN) until ∼70% and then rises sharply to ∼23 pN at high filling, which is a several-fold lower value than was previously estimated under the assumption that force alone slows the motor. These findings are consistent with recent studies of the stepping kinetics of the motor. The allosteric regulatory mechanism we report allows double-stranded DNA viruses to achieve rapid, high-density packing of their genomes by limiting the buildup of nonequilibrium load forces on the motor.

In Chapter IV I attempt to synthesize the previous chapters into a more unified picture and reflect on what I learned and the implications of our findings. I also discuss a few of the numerous projects that can expand upon the work already completed here, focusing particularly on using DNA ejection measurements to study DNA relaxation on much longer timescale than would be possible with single molecule experiments.

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