A DNA packaging motor inchworms along one strand allowing it to adapt to alternative double-helical structures.

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.

Using a system's equilibrium behavior to reduce its energy dissipation in nonequilibrium processes.

Cells must operate far from equilibrium, utilizing and dissipating energy continuously to maintain their organization and to avoid stasis and death. However, they must also avoid unnecessary waste of energy. Recent studies have revealed that molecular machines are extremely efficient thermodynamically compared with their macroscopic counterparts. However, the principles governing the efficient out-of-equilibrium operation of molecular machines remain a mystery. A theoretical framework has been recently formulated in which a generalized friction coefficient quantifies the energetic efficiency in nonequilibrium processes. Moreover, it posits that, to minimize energy dissipation, external control should drive the system along the reaction coordinate with a speed inversely proportional to the square root of that friction coefficient. Here, we demonstrate the utility of this theory for designing and understanding energetically efficient nonequilibrium processes through the unfolding and folding of single DNA hairpins.

Molecular switch-like regulation enables global subunit coordination in a viral ring ATPase.

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.

Molecular switch-like regulation in motor proteins.

Motor proteins are powered by nucleotide hydrolysis and exert mechanical work to carry out many fundamental biological tasks. To ensure their correct and efficient performance, the motors' activities are allosterically regulated by additional factors that enhance or suppress their NTPase activity. Here, we review two highly conserved mechanisms of ATP hydrolysis activation and repression operating in motor proteins-the glutamate switch and the arginine finger-and their associated regulatory factors. We examine the implications of these regulatory mechanisms in proteins that are formed by multiple ATPase subunits. We argue that the regulatory mechanisms employed by motor proteins display features similar to those described in small GTPases, which require external regulatory elements, such as dissociation inhibitors, exchange factors and activating proteins, to switch the protein's function 'on' and 'off'. Likewise, similar regulatory roles are taken on by the motor's substrate, additional binding factors, and even adjacent subunits in multimeric complexes. However, in motor proteins, more than one regulatory factor and the two mechanisms described here often underlie the machine's operation. Furthermore, ATPase regulation takes place throughout the motor's cycle, which enables a more complex function than the binary 'active' and 'inactive' states.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

Deciphering the Molecular Mechanism of the Bacteriophage φ29 DNA Packaging Motor.

The past decade has seen an explosion in the use of single-molecule approaches to study complex biological processes. One such approach-optical trapping-is particularly well suited for investigating molecular motors, a diverse group of macromolecular complexes that convert chemical energy into mechanical work, thus playing key roles in virtually every aspect of cellular life. Here we describe how to use high-resolution optical tweezers to investigate the mechanism of the bacteriophage φ29 DNA packaging motor, a ring-shaped ATPase responsible for genome packing during viral assembly. This system illustrates how to use single-molecule techniques to uncover novel, often unexpected, principles of motor operation.

A viral packaging motor varies its DNA rotation and step size to preserve subunit coordination as the capsid fills.

Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per base pair increases with filling. This change accompanies a reduction in the motor's step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the downregulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated.