Dynamics of Viral Packaging: Single-Molecule Observations in Multiple Dimensions
- Author(s): Hetherington, Craig Lee
- Advisor(s): Bustamante, Carlos J
- et al.
During the self-assembly of bacteriophage phi29, the viral genome is packaged into a pre-formed capsid by a molecular motor. The packaging motor is a complex of several oligomers including a dodecameric connector ring and a pentameric ATPase ring. These rings coordinate with each other, generating high forces in order to compact the dsDNA genome into the capsid at high pressures.
The connector was proposed to engage and directly perform work on the DNA during packaging. Consideration of the symmetry of the connector and the capsid predicts that the connector must rotate relative to the capsid as part of the mechanism for translocating the DNA. An experiment designed to directly measure rotation of the connector is discussed in Chapter 2 of this dissertation. A combination magnetic tweezers and total internal reflection microscope is used to track the polarization of single fluorescent dyes attached to the connector. No evidence of polarization changes were found, indicating that the connector does not rotate at the expected rates. This further suggests that the connector does not directly perform mechanical work on the DNA during packaging.
Viral packaging can be observed in optical tweezers by monitoring the length of the DNA as it is drawn into the capsid. Past studies have revealed many details of the packaging mechanism by following the dependence of the packaging velocity on factors like ATP concentration and applied load. In Chapter 3, I propose an experimental design intended to measure the effect of the packaging motor on the angle of the DNA in addition to its length and thus recover the full three-dimensional trajectory of the DNA as it passes into the capsid. In addition, this scheme can be used to apply torque and thus provides an additional tool with which to probe the packaging mechanism.
In Chapter 4, we find that during packaging the downstream DNA is twisted in an underwinding direction, with a magnitude that depends on the extent to which the capsid is filled. The change in twisting can be attributed to cumulative looping of the DNA within the capsid, and the data predicts that the loops formed in the last kilobasepair of packaging are as small as 4 nm in radius. In addition, a non-lethal method of rupturing the viral capsids prior to packaging was discovered. Observations of DNA twisting by those packaging complexes revealed that, in the absence of internal pressure, the DNA is twisted by -1.2 °/bp. This number suggests that one of the packaging motor's five subunits makes contact with the DNA every ten basepairs, and that the cycle repeats with that subunit performing the same function every time. Such a strict functional segregation, in addition to the catalytic segregation revealed in previous high-resolution optical tweezers experiments, is an important part of the mechanism by which the motor packages DNA against high forces.