Abstract: 

Many transport processes in the cell are performed by a diverse but structurally and functionally related family of proteins. These proteins, which belong to the ASCE (Additional Strand, Conserved E) superfamily of ATPases, often form mutimeric rings.  Despite their importance, a number of fundamental questions remain as to the coordination of the various subunits in these rings. Bacteriophage phi29 packages its 6.6 mm long double-stranded DNA using a pentameric ring nano motor Using optical tweezers, we find that this motor can work against loads of up to ~55 picoNewtons on average, making it one of the strongest molecular motors ever reported. Interestingly, the packaging rate decreases as the prohead fills, indicating that an internal pressure builds up due to DNA compression attaining the value of ~3 MegaPascals at the end of packaging, a pressure that is used as part of the mechanism of DNA injection in the next infection cycle. We have used high-resolution optical tweezers to show that the motor packages the DNA in alternating phases of dwells and bursts. During the dwell the motor exchanges nucleotide, whereas during the burst, the motor packages 10 bps of DNA per cycle.  We have also characterized the steps and intersubunit coordination of this ATPase. By using non-hydrolyzable ATP analogs and stabilizers of the ADP bound to the motor, we establish where DNA binding, hydrolysis, and phosphate and ADP release occur relative to translocation during the motor’s cycle.  Surprisingly, a division of labor exists among the subunits: while only 4 of the subunits translocate DNA, all 5 bind and hydrolyze ATP, suggesting that the fifth subunit fulfills a regulatory function. Furthermore, we show that the motor not only can generate force but also torque. We characterize the role played by the special subunit in this process and identify the symmetry-breaking mechanism in the motor. Finally, we use dsRNA, and RNA/DNA hybrids to show that the size of the burst phase adapts to the periodicity of the substrate being translocated. This information, together with recent structural data, allows us to propose a novel mechanism of translocation for this motor.

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