Single-Molecule Measurements of Motor-Driven Viral DNA Packaging in Bacteriophages Phi29, Lambda, and T4 with Optical Tweezers.
Viral DNA packaging is a required step in the assembly of many dsDNA viruses. A molecular motor fueled by ATP hydrolysis packages the viral genome to near crystalline density inside a preformed prohead shell in ~5 min at room temperature. We describe procedures for measuring the packaging of single DNA molecules into single viral proheads with optical tweezers. Three viral packaging systems are described in detail: bacteriophages phi29 (φ29), lambda (λ), and T4. Two different approaches are described: (1) With φ29 and T4, prohead-motor complexes can be preassembled in bulk and packaging can be initiated in the optical tweezers by "feeding" a single DNA molecule to one of the complexes; (2) With φ29 and λ, packaging can be initiated in bulk then stalled, and a single prohead-motor-DNA complex can then be captured with optical tweezers and restarted. In both cases, the prohead is ultimately attached to one trapped microsphere and the end of the DNA being packaged is attached to a second trapped microsphere such that packaging of the DNA pulls the two microspheres together and the rate of packaging and force generated by the motor is directly measured in real time. These protocols allow for the effect of many experimental parameters on packaging dynamics to be studied such as temperature, ATP concentration, ionic conditions, structural changes to the DNA substrate, and mutations in the motor proteins. Procedures for capturing microspheres with the optical traps and different measurement modes are also described.
['Keller|Nicholas|N|', 'delToro|Damian J|DJ|', 'Smith|Douglas E|DE|']
D017122:Bacteriophage T4 D010582:Bacteriophage lambda D001435:Bacteriophages D019921:Biotinylation D042003:DNA Packaging D004279:DNA, Viral D008863:Microspheres D020409:Molecular Motor Proteins D052898:Optical Tweezers D016133:Polymerase Chain Reaction D000072760:Single Molecule Imaging D019065:Virus Assembly
Viral DNA packaging Optical tweezers Molecular motor Single-molecule manipulation