Analyzing DNA Packaging Initiation of Bacteriophage T4 by a Real-Time Single Molecule Fluorescence Assay

Variety of structural, biochemical and recently single molecule optical tweezers experiments have been used to study the mechanochemical aspects of viral packaging motors. However, due to transient and complex nature of interactions, it has been difficult to quantify viral assembly and packaging initiation using these techniques. We developed a single molecule fluorescence assay to study initiation and re-initiation of DNA packaging in the T4 bacteriophage. The reconstituted complexes were immobilized on a polymer covered slide and fluorescently labeled, short double stranded DNA and ATP were added and individual packaging machines, each carrying out DNA translocation, were imaged in real-time by total internal reflection microscopy. We found that the T4 packaging machine can package multiple DNA into the same head with the re-initiation rate depending on the DNA and ATP concentrations. Mutations in the Walker A P-loop of the packaging motor showed very low initiation efficiency, slow initiation time, and far fewer initiations per head. By quantifying the assembly efficiency in the presence of different co-factors, we found that while the motor is stable in the ATP-bound state after ATP hydrolysis the motor is much less stably bound to the viral capsid. The presence of a DNA in the portal channel increases the stability of motor complex. This assay can be extended to study the effect of other relevant proteins on the assembly and initiation of DNA packaging.