Single phage T4 DNA packaging motors exhibit large force generation, high velocity, and dynamic variability

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101

Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate. Here, we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.soft matter | virology | DNA condensation D NA packaging is both a critical step in viral assembly and a unique model for understanding the physics of polymers under strong confinement. Before packaging, the DNA (∼6-60 μm long) forms a loose random coil of diameter ∼1-3 μm. After translocation into the viral prohead (∼50-100 nm in diameter), a ∼10,000-fold volume compaction is achieved. Packaging is driven by a powerful molecular motor that must work against the large forces resisting confinement arising from DNA bending, repulsion between DNA segments, and entropy loss (1-8).DNA packaging in bacteriophages phi29, lambda, and T4 has been directly measured via single-molecule manipulation with optical tweezers and the packaging motors have been shown to generate forces of >60 pN, among the highest known for biomotors, while translocating DNA at rates ranging from ∼100 bp (for phage phi29, which packages a 19.3-kbp genome into a 42 × 54-nm prohead shell) up to as high as ∼2,000 bp/s (for phage T4, which packages a 171-kbp genome into a 120 × 86-nm prohead) (9-15). The force resisting packaging rises steeply with prohead filling and has been proposed to play an important role in driving viral DNA ejection (16).Recently, a variety of theoretical models for viral DNA packaging have been proposed (3)(4)(5)(17)(18)(19)(20)(21). The simplest treat DNA as an elastic rod with repulsive self-interactions and assume that packaging is a quasistatic thermodynamic process, i.e., that the DNA is able to continuously relax to a free-energy minimum state (3)(4)(5)(19)(20)(21). The DNA arrangement is generally assumed ...

Section: Significancementioning

confidence: 99%

mentioning

confidence: 99%

Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging

Berndsen

Keller²,

Grimes

et al. 2014

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101

“…Our previous optical tweezers assays with WT motor complexes revealed that packaging could be triggered to occur within seconds of bringing a target DNA molecule into contact with a procapsidterminase complex and that packaging at a high speed averaging 580 bp/s usually proceeded within seconds (8). Similar optical tweezers assays were previously applied to measuring packaging dynamics in bacteriophage phi29 and have also recently been applied to bacteriophage T4 (9). These optical tweezers assays also revealed that all of these viral motors were very strong, exerting Ͼ50 pN of force during packaging.…”

mentioning

confidence: 96%

The Q motif of a viral packaging motor governs its force generation and communicates ATP recognition to DNA interaction

Tsay

Sippy

Feiss

et al. 2009

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A key step in the assembly of many viruses is the packaging of DNA into preformed procapsids by an ATP-powered molecular motor. To shed light on the motor mechanism we used single-molecule optical tweezers measurements to study the effect of mutations in the large terminase subunit in bacteriophage on packaging motor dynamics. A mutation, K84A, in the putative ATPase domain driving DNA translocation was found to decrease motor velocity by Ϸ40% but did not change the force dependence or decrease processivity substantially. These findings support the hypothesis that a deviant ''Walker A-like'' phosphate-binding motif lies adjacent to residue 84. Another mutation, Y46F, was also found to decrease motor velocity by Ϸ40% but also increase slipping during DNA translocation by >10-fold. These findings support the hypothesis that viral DNA packaging motors contain an adeninebinding motif that regulates ATP hydrolysis and substrate affinity analogous to the ''Q motif'' recently identified in DEAD-box RNA helicases. We also find impaired force generation for the Y46F mutant, which shows that the Q motif plays an important role in determining the power and efficiency of the packaging motor.optical tweezers ͉ phage ͉ single molecule ͉ virus

“…Structural studies (12,16,19) and single-molecule optical tweezers (3,13) and fluorescence spectroscopy (20,21) approaches have been used to dissect the mechanochemical steps of DNA translocation. However, the transient nature of DNA and protein interactions at the initiation stage, which involve insertion of the dsDNA end into the prohead and triggering of translocation, has been a major challenge (22).…”

Section: Significancementioning

confidence: 99%

“…The T4 packaging motor, a pentamer of gp17 (70 kDa) (large terminase protein) assembled on the gp20 portal dodecamer (12) is the fastest (packaging rate up to ∼2,000 bp/s) of the viral packaging motors studied (13). Gp17 possesses all of the basic enzymatic activities necessary for generating a DNA-full head: ATPase, nuclease, and translocase (14,15).…”

mentioning

confidence: 99%

Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4

Vafabakhsh

Kondabagil

Earnest

et al. 2014

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Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and singlemolecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.virus packaging | single-molecule fluorescence imaging | molecular motors A s part of a virus life cycle, genetic information needs to be incorporated into the newly produced virus particles. Tailed bacteriophages, which probably form the largest biomass of the planet (1), and many eukaryotic viruses such as herpes viruses use powerful ATPase motors to achieve this (2). These motors generate forces as high as 80-100 pN and translocate DNA into a preformed prohead until a DNA condensate of near crystalline density fills the interior (3).The viral packaging motors share a common architecture with the ASCE (additional strand, conserved E) superfamily of multimeric ring ATPases that perform diverse functions such as chromosome segregation (helicases), protein remodeling (chaperones and proteasomes), and cargo transport (dyneins) (4). Although much has been learned about the mechanochemistry of these motors, little is known about how a functional motor is assembled and its activity is initiated. The packaging motors have the difficult task of precisely inserting the end of a viral genome into the capsid at the time of initiation.In a general virus assembly pathway shared by dsDNA viruses, assembly starts at a unique fivefold vertex of the prohead called the portal vertex, which is formed from 12 molecules of the portal protein (5). A protein shell assembles around a protein scaffold and later becomes an empty prohead after the scaffold leaves, or is degraded (6). In most dsDNA bacteriophages as well as herpes viruses a complex of two proteins, known as small and large "terminase" proteins, recognize a specific sequence of DNA in the concatemeric viral genome (e.g., cos site in phage λ and pac site in phage P22) and make a cut to create a dsDNA end (7,8). The small terminase is responsible for binding to the cos or pac site, whereas the large terminase makes the cut. However, phage phi29 and adenoviruses do not require DNA cutting because the genome is a unit-length m...