Mechanism of Coordination of the Bacteriophage T4 DNA Packaging Motor Analyzed by Real-Time Single Molecule Fluorescence Assay

Abstract

Bacteriophage T4 packages its DNA genome into the viral capsid using a powerful molecular motor. The motor is found to be a pentamer by cryo-EM. However, the stoichiometry of the actively packaging motor is unknown. Structural and biochemical studies suggest that gp17 converts energy from ATP hydrolysis into electrostatic force which drives DNA translocation. Very little is known about how the motor subunits coordinate to package DNA at a rate of up to ∼2000 bp/s, the fastest rate reported to date.By reconstituting the functional T4 packaging complex with Cy3-labeled gp17 and incubating it with Cy5-DNA, each motor carrying out translocation of Cy5-DNA was imaged in real-time using total internal reflection fluorescence (TIRF) microscopy. Simultaneous imaging of the Cy3-gp17 subunits of the packaging complex and Cy5-DNA being packaged allowed us to determine motor stoichiometry by direct counting of the number of photobleaching steps. These data showed that the motor complex contains five gp17 subunits, but not six. Thus the stoichiometry of the actively packaging motor is a pentamer.Cy3-label was introduced into a mutant gp17 subunit, Q163A-gp17, which retained the ability to assemble motors with the wild-type gp17 subunits but is inactive for DNA packaging because its ATPase function is defective. Analysis of single packaging complexes containing 0 to 5 mutant subunits showed that motors containing 1 or 2 mutant subunits can package DNA. However, the rate of initiation and efficiency of DNA packaging were reduced.We conclude that the pentameric T4 packaging motor is not strictly coordinated. If it were, packaging would have been stalled at the inactive subunit. It appears that when an inactive subunit is encountered, the motor pauses transiently, then recovers and switches to another subunit and continues translocation.