Molecular Dynamics of DNA Translocation by FtsK

The bacterial FtsK motor harvests energy from ATP to translocate double-stranded DNA during cell division. Here, we probe the molecular mechanisms underlying coordinated DNA translocation in FtsK by performing long timescale simulations of its hexameric assembly and individual subunits. From these simulations we predict signaling pathways that connect the ATPase active site to DNA-gripping residues, which allows the motor to coordinate its translocation activity with its ATPase activity. Additionally, we utilize well-tempered metadynamics simulations to compute free-energy landscapes that elucidate the extended-to-compact transition involved in force generation. We show that nucleotide binding promotes a compact conformation of a motor subunit, whereas the apo subunit is flexible. Together, our results support a mechanism whereby each ATP-bound subunit of the motor conforms to the helical pitch of DNA, and ATP hydrolysis/product release causes a subunit to lose grip of DNA. By ordinally engaging and disengaging with DNA, the FtsK motor unidirectionally translocates DNA.

A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

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.

Electronic structure of molecular hydrogen in MoS2 nanopores

Atom controlled sub-nanometer MoS2 pores have been recently fabricated with promising applications, such gas sensing, hydrogen storage and DNA translocation. In this work we carried out first-principles calculations of hydrogen adsorption in tiny MoS2 nanopores. Some of the pores show metallic behavior whereas others have a sizeable band gap. Whereas adsorption of molecular hydrogen on bare pores are dominated by physisorption, adsorption in the nanopores show chemisorption behavior with high selectivity depending on the pore inner termination. Finally, we show that functionalization with copper atoms leads to does not improve dignificantly the adsorption energies of selected pores.selected pores.

Membrane fission during bacterial spore development requires DNA-driven cellular inflation

Bacteria require membrane fission for cell division and endospore formation. FisB catalyzes membrane fission during sporulation, but the molecular basis is unclear as it cannot remodel membranes by itself. Sporulation initiates with an asymmetric division that generates a large mother cell and a smaller forespore that contains only 1/4 of its complete genome. As the mother cell membranes engulf the forespore, a DNA translocase pumps the rest of the chromosome into the small forespore compartment, inflating it due to increased turgor. When the engulfing membranes undergo fission, the forespore is released into the mother cell cytoplasm. Here we show that forespore inflation and FisB accumulation are both required for efficient membrane fission. We suggest that high membrane tension in the engulfment membrane caused by forespore inflation drives FisB-catalyzed membrane fission. Collectively our data indicate that DNA-translocation has a previously unappreciated second function in energizing FisB-mediated membrane fission under energy-limited conditions.

Fast Fabrication of Solid-State Nanopores for DNA Molecule Analysis

Solid-state nanopores have been developed as a prominent tool for single molecule analysis in versatile applications. Although controlled dielectric breakdown (CDB) is the most accessible method for a single nanopore fabrication, it is still necessary to improve the fabrication efficiency and avoid the generation of multiple nanopores. In this work, we treated the SiNx membranes in the air–plasma before the CDB process, which shortened the time-to-pore-formation by orders of magnitude. λ-DNA translocation experiments validated the functionality of the pore and substantiated the presence of only a single pore on the membrane. Our fabricated pore could also be successfully used to detect short single-stranded DNA (ssDNA) fragments. Using to ionic current signals, ssDNA fragments with different lengths could be clearly distinguished. These results will provide a valuable reference for the nanopore fabrication and DNA analysis.

Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope

In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the Podoviridae family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects Escherichia coli, and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15–gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.

Stochastic Resonance Behavior of DNA Translocation with an Oscillatory Electric Field

Stochastic resonance (SR) describes the synchronization between noise of a system and an applied oscillating field to achieve an optimized response signal. In this work, we use simulations to investigate the phenomenon of SR of a single stranded DNA driven through a nanopore when an oscillating electric field is added. The system is comprised of a MspA protein nanopore embedded in a membrane and different lengths of DNA is driven from one end of the pore to the other via a constant potential difference. We superimposed an oscillating electric field on top of the existing electric field. The source of noise is due to thermal fluctuations, since the system is immersed in solution at room temperature. Here, the signal optimization we seek is the increase in translocation time of DNA through the protein nanopore. Normally, translocation time scales linearly with DNA length and inversely with driving force in a drift dominanted regime. We found a non monotonic dependence of the mean translocation time with the frequency of the oscillating field. This non-monotonic behavior of the translocation time is observed for all lengths of DNA, but SR occurs only for longer DNA. Furthermore, we also see evidence of DNA extension being influenced by the oscillating field while moving through the nanopore.