The bacteriophage φ29 portal motor can package DNA against a large internal force (original) (raw)
Related papers
Cell, 2008
Viral genomes are packaged into ''procapsids'' by powerful molecular motors. We report the crystal structure of the DNA packaging motor protein, gene product 17 (gp17), in bacteriophage T4. The structure consists of an N-terminal ATPase domain, which provides energy for compacting DNA, and a C-terminal nuclease domain, which terminates packaging. We show that another function of the C-terminal domain is to translocate the genome into the procapsid. The two domains are in close contact in the crystal structure, representing a ''tensed state.'' A cryo-electron microscopy reconstruction of the T4 procapsid complexed with gp17 shows that the packaging motor is a pentamer and that the domains within each monomer are spatially separated, representing a ''relaxed state.'' These structures suggest a mechanism, supported by mutational and other data, in which electrostatic forces drive the DNA packaging by alternating between tensed and relaxed states. Similar mechanisms may occur in other molecular motors.
DNA Packaging Motor Assembly Intermediate of Bacteriophage ϕ29
Journal of Molecular Biology, 2008
Unraveling the structure and assembly of the DNA packaging ATPases of the tailed, double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here the bacteriophage ϕ29 packaging ATPase, gene product gp16 (gp16), was over-expressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the ϕ29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent upon the presence of an oligomer of 174-or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18-20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone, and therefore the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway.
Headful DNA packaging: bacteriophage SPP1 as a model system
Virus research, 2013
Tailed bacteriophages and herpesviruses package DNA inside the viral capsid by a powerful molecular motor. This packaging machine is composed of the portal protein, which provides a gate for DNA entry, the large terminase subunit whose ATPase activity fuels DNA translocation, and most frequently, a small terminase subunit that recognizes the viral packaging site. Here we review the mechanisms how the virulent Bacillus subtilis phage SPP1 packages DNA into a preformed procapsid. Encapsidation of the SPP1 DNA follows a processive unidirectional headful mechanism that starts with the recognition and cleavage of a unique genomic sequence (pac) by the viral terminase. The viral genome is then translocated through the central channel of the portal protein found at a single vertex of the procapsid. Packaging is terminated by an endonucleolytic cleavage of the concatemeric DNA substrate, following by disassembly of the packaging motor and closure of the portal system by the gatekeepers prev...
The Bacteriophage DNA Packaging Motor
Annual Review of Genetics, 2008
An ATP-powered DNA translocation machine encapsidates the viral genome in the large dsDNA bacteriophages. The essential components include the empty shell, prohead, and the packaging enzyme, terminase. During translocation, terminase is docked on the prohead's portal protein. The translocation ATPase and the concatemer-cutting endonuclease reside in terminase. Remarkably, terminases, portal proteins, and shells of tailed bacteriophages and herpes viruses show conserved features. These DNA viruses may have descended from a common ancestor. Terminase's ATPase consists of a classic nucleotide binding fold, most closely resembling that of monomeric helicases. Intriguing models have been proposed for the mechanism of dsDNA translocation, invoking ATP hydrolysis-driven conformational changes of portal or terminase powering DNA motion. Single-molecule studies show that the packaging motor is fast and powerful. Recent advances permit experiments that can critically test the packaging models. The viral genome translocation mechanism is of general interest, given the parallels between terminases, helicases, and other motor proteins.
A Promiscuous DNA Packaging Machine from Bacteriophage T4
PLOS Biology, 2011
Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.
Mechanics of DNA packaging in viruses
Proceedings of The National Academy of Sciences, 2003
A new generation of single-molecule experiments has opened up the possibility of reexamining many of the fundamental processes of biochemistry and molecular biology from a unique and quantitative perspective. One technique producing a host of intriguing results is the use of optical tweezers to measure the mechanical forces exerted by molecular motors during key processes such as the transcription of DNA or the packing of a viral genome into its capsid. The objective of the current article is to respond to such measurements on viruses and to use the theory of elasticity and a simple model of charge and hydration forces to derive the force required to pack DNA into a viral capsid as a function of the fraction of the viral genome that has been packed. The results are found to be in excellent accord with recent measurements and complement previous theoretical work. Because the packing of DNA in viral capsids occurs under circumstances of high internal pressure, we also compute how much pressure a capsid can sustain without rupture. I n rapid succession over the last several years, a number of new experimental insights into the way DNA in viruses is packaged and ejected have been garnered. For example, the structure of both the portal motor (1) as well as an example of the membrane puncturing device that leads to the delivery of the viral genome have been determined (2). At lower resolution, results of cryoelectron microscopy experiments have revealed the structure of certain viruses at various stages during self-assembly (3) and the ordered arrangements of DNA in concentric circles within viral capsids (4-6). These insights have recently been complemented by single-molecule experiments in which the force exerted by the portal motor is measured during the process of viral packing itself .
Cell, 2014
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.
Substrate interactions and promiscuity in a viral DNA packaging motor
Nature, 2009
The ASCE (additional strand, conserved E) superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life. A subset of these enzymes consists of multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses and tailed bacteriophages. Although their mechanism of mechanochemical conversion is beginning to be understood, little is known about how these motors engage their nucleic acid substrates. Questions remain as to whether the motors contact a single DNA element, such as a phosphate or a base, or whether contacts are distributed over several parts of the DNA. Furthermore, the role of these contacts in the mechanochemical cycle is unknown. Here we use the genome packaging motor of the Bacillus subtilis bacteriophage varphi29 (ref. 4) to address these questions. The full mechanoche...
Nature Communications
ATP-powered viral packaging motors are among the most powerful biomotors known. Motor subunits arranged in a ring repeatedly grip and translocate the DNA to package viral genomes into capsids. Here, we use single DNA manipulation and rapid solution exchange to quantify how nucleotide binding regulates interactions between the bacteriophage T4 motor and DNA substrate. With no nucleotides, there is virtually no gripping and rapid slipping occurs with only minimal friction resisting. In contrast, binding of an ATP analog engages nearly continuous gripping. Occasional slips occur due to dissociation of the analog from a gripping motor subunit, or force-induced rupture of grip, but multiple other analog-bound subunits exert high friction that limits slipping. ADP induces comparably infrequent gripping and variable friction. Independent of nucleotides, slipping arrests when the end of the DNA is about to exit the capsid. This end-clamp mechanism increases the efficiency of packaging by making it essentially irreversible.