Ring ATPases that translocate disordered polymers possess lock-washer architectures that they impose on their substrates during transport via a hand-over-hand mechanism. Here, we investigate the operation of ring motors that transport ordered, helical substrates, such as the bacteriophage ϕ29 dsDNA packaging motor. This pentameric motor alternates between an ATP loading dwell and a hydrolysis burst wherein it packages one turn of DNA in four steps. When challenged with DNA-RNA hybrids and dsRNA, the motor matches its burst to the shorter helical pitches, keeping three power strokes invariant while shortening the fourth. Intermittently, the motor loses grip on the RNA-containing substrates, indicating that it makes optimal load-bearing contacts with dsDNA. To rationalize these observations, we propose a helical inchworm translocation mechanism in which, during each cycle, the motor increasingly adopts a lock-washer structure during the ATP loading dwell and successively regains its planar form with each power stroke during the burst.

Genetic and physiological studies of dsDNA bacteriophages have established their assembly pathways in some detail, yet the precise mechanism of DNA packaging remains obscure. We follow the packaging process step-wise, from the initial state in which an empty capsid engages a free, extended genome in solution, to the final state, in which 100% of the genome is tightly packed inside the capsid. Our work is aimed at understanding details of the DNA packaging process. Force measurements with optical tweezers provide values of the pressure generated inside the capsid by the DNA, as a function of the percentage of the total genome already packaged. Cryo-EM provides a direct visualization of the architecture of the DNA inside the capsid and thus direct measurements of fundamental physical parameters, such as inter-strand distances, local curvatures, and local and long range degree of order. Using Monte Carlo simulations, we predict the conformation of the DNA in the interior of the viral capsid and the forces required to achieve this structure. The resulting density plots from simulations show a qualitatively good agreement with cryo-EM image analysis of bacteriophage with partially packaged genomes; the predicted forces are consistent with previous single-molecule measurements of the packaging forces, although small quantitative differences remain, and current work aims at obtaining a better agreement. Conformations from the Monte Carlo simulations tend to exhibit local layer ordering with frequent defects associated with chain segments jumping between layers. We discuss the impact that such defects have on the overall order and the resistance to DNA packaging and ejection.

Subunits in multimeric ring-shaped motors must coordinate their activities to ensure correct and efficient performance of their mechanical tasks. Here, we study WT and arginine finger mutants of the pentameric bacteriophage φ29 DNA packaging motor. Our results reveal the molecular interactions necessary for the coordination of ADP-ATP exchange and ATP hydrolysis of the motor's biphasic mechanochemical cycle. We show that two distinct regulatory mechanisms determine this coordination. In the first mechanism, the DNA up-regulates a single subunit's catalytic activity, transforming it into a global regulator that initiates the nucleotide exchange phase and the hydrolysis phase. In the second, an arginine finger in each subunit promotes ADP-ATP exchange and ATP hydrolysis of its neighbor. Accordingly, we suggest that the subunits perform the roles described for GDP exchange factors and GTPase-activating proteins observed in small GTPases. We propose that these mechanisms are fundamental to intersubunit coordination and are likely present in other ring ATPases.

Genetic and physiological studies of dsDNA bacteriophage have established their assembly pathways in some detail, yet the precise mechanism of DNA packaging remains obscure. We combine two powerful techniques with the aim of further elucidating the mechanism of DNA packaging and ejection in bacteriophage: three-dimensional cryo-EM imaging to obtain precise structural measurements and optical tweezers to obtain direct measurements of the forces involved. We follow the process step-wise, from the initial state in which an empty capside engages a free, extended genome in solution, to the final state, in which 100 percent of the genome is tightly packed inside the capside. Current work has provided strikingly beautiful three-dimensional reconstructions of whole bacteriophage in which the ordered structure of the genome can be clearly appreciated. However, in these works, the DNA is averaged out of register from particle to particle, resulting in sets of quite perfect and independent rings. This could be a consequence of the effort to obtain a high resolution reconstruction of all protein components. The pitch and the threading of the dsDNA are lost, as is any idea of its actual trajectory throughout the packaging process. Alternatively, another possibility is that the DNA occupies the interior of the capside following a highly ordered overall architecture, but with a considerable degree of local disorder and defects. Our work aims at elucidating some of these DNA-specific details. Force measurements with optical tweezers provide the pressure generated inside the capside by the DNA, as a function of the percentage of the total genome already packaged. Cryo-EM provides a direct visualization of the architecture of the DNA inside the capsid and thus direct measurements of fundamental physical parameters, such as inter-strand distances, local curvatures, and local and long range degree of order. Using Monte Carlo simulations, we predict the conformation of the DNA on the interior of the viral capsid and the forces required to achieve this structure. The resulting density plots from simulations show a good qualitatively agreement with cryo-EM results; the predicted forces are consistent with previous single-molecule measurements of the packaging forces, although small quantitative differences remain, and current work aims at obtaining a better agreement. Conformations from the Monte Carlo simulations tend to exhibit local layer ordering with frequent defects associated with chain segments jumping across layers. We discuss the impact that such defects have on the overall order and the resistance to DNA packaging and ejection.