| Bacteriophage T4 packages its double-stranded DNA genome into a proteinaceous capsid using a pentameric gp17 motor assembled at the special dodecameric portal vertex. This complex is referred to as the DNA packaging machine. Previous analysis of single DNA packaging machines using dual optical tweezers showed that the T4 motor is one of the fastest and most powerful reported to date. Structural and biochemical studies suggested that the motor converts chemical energy into electrostatic force, which then drives DNA translocation. Although the structure and function of this molecular motor have been well-established, several basic questions on the motor mechanism remain poorly understood. Cryo-EM reconstruction showed five subunits of gp17 bound to the portal vertex, but it is not clear whether this is also true for machines that actively package DNA. Are the motor subunits coordinated during translocation? Is there a sequential order by which the subunits fire ATP hydrolysis?;We analyzed T4 DNA packaging using a real-time single molecule fluorescence assay to quantitatively analyze the stoichiometry and coordination of actively packaging machines. By attaching the Cy3 fluorescent label to the motor subunit and direct counting, we establish that the actively packaging T4 motor is a pentamer. We then created mutant motors containing one or more Cy3-labeled inactive ("dead") subunits and observed the packaging of Cy5-labeled DNA in real-time. We found that the T4 packaging motors containing one dead subunit can still initiate packaging and translocate DNA. However, the mutant motor packaged fewer molecules of DNA into the head and its initiation time was slower than the wild-type motor. These results do not fit with the behavior of the classic ring-type helicase motors or the phage phi29 DNA packaging motor, which are strictly coordinated and sequential. The T4 motor appears to be a new type of motor, a "plastic" motor that can tolerate a defective subunit. It is not strictly coordinated. It appears that each subunit fires independently of other subunits, and when a defective subunit is encountered, the motor pauses transiently, hands over the DNA to an adjacent subunit, and continues translocation. |
