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Abstract Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, using single-molecule fluorescence, we determine that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise number of active or inactive subunits, we find that the packaging motor can tolerate an inactive subunit. However, motors containing one or more inactive subunits exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a DNA packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, suggesting that strict coordination amongst motor subunits of packaging motors is not crucial for function. 

The inside of a cell is a crowded space, full of proteins and other molecules. Yet, the molecular motors that transport some of those molecules within the cell move at the same speed as they would in pure water – about one micrometer per second. How the molecular motors could achieve such speeds in crowded cells was unclear. Nevertheless, Tjioe et al. suspected that the answer might be related to how multiple motors work together. Molecular motors move by walking along filaments inside the cell and pulling their cargo from one location to another. Other molecules that bind to the filaments should, in theory, act like “roadblocks” and impede the movement of the cargo. Tjioe et al. studied a motor protein called kinesin, which walks on filaments called microtubules. But instead of looking at these motors moving along microtubules inside a cell, Tjioe et al. used a simpler system where the cell was eliminated, and all parts were purified. Specifically, Tjioe et al. tethered purified motors to a piece of glass and then observed them under an extremely accurate microscope as they moved free-floating, fluorescently labelled microtubules. The microtubules, in this scenario, were acting like cargoes, where many kinesins could bind. Each kinesin motor also had a small chemical tag that could emit light. By following the movement of the lights, it was possible to calculate what each kinesin was doing and how the cargo moved. When more than one kinesin molecule was acting, the tension and speed of one kinesin affected the movement of the others. In any group of kinesins, about two-thirds of kinesin pulled the cargo, and unexpectedly, about one-third tended to resist and slow the cargo. These latter kinesins were moved along with the group without actually driving the cargo. These resisting kinesins did come off more rapidly than the driving kinesins, meaning the cargo should be able to quickly bypass roadblocks. This would help to keep the whole group travelling in the right direction at a steady pace. 

Helicases play key roles in genome maintenance, yet it remains elusive how these enzymes change conformations and how transitions between different conformational states regulate nucleic acid reshaping. Here, we developed a computational technique combining structural bioinformatics approaches and atomic-level free-energy simulations to characterize how the Escherichia coli DNA repair enzyme UvrD changes its conformation at the fork junction to switch its function from unwinding to rezipping DNA. The lowest free-energy path shows that UvrD opens the interface between two domains, allowing the bound ssDNA to escape. The simulation results predict a key metastable 'tilted' state during ssDNA strand switching. By simulating FRET distributions with fluorophores attached to UvrD, we show that the new state is supported quantitatively by single-molecule measurements. The present study deciphers key elements for the 'hyper-helicase' behavior of a mutant and provides an effective framework to characterize directly structure-function relationships in molecular machines.