Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes.

The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.

Structural changes in bacteriophage T7 upon receptor-induced genome ejection.

Many tailed bacteriophages assemble ejection proteins and a portal-tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel extending the portal-tail channel for the delivery of genomic DNA in cell infection. Here, we report the structure of the mature bacteriophage T7, including the ejection proteins, as well as the structures of the full and empty T7 particles in complex with their cell receptor lipopolysaccharide. Our near-atomic-resolution reconstruction shows that the ejection proteins in the mature T7 assemble into a core, which comprises a fourfold gene product 16 (gp16) ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a lytic transglycosylase domain for degrading the bacterial peptidoglycan layer. When interacting with the lipopolysaccharide, the T7 tail nozzle opens. Six copies of gp14 anchor to the tail nozzle, extending the nozzle across the lipopolysaccharide lipid bilayer. The structures of gp15 and gp16 in the mature T7 suggest that they should undergo remarkable conformational changes to form the transenvelope channel. Hydrophobic α-helices were observed in gp16 but not in gp15, suggesting that gp15 forms the channel in the hydrophilic periplasm and gp16 forms the channel in the cytoplasmic membrane.

Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis.

We present a structure of the ∼650-kDa functional replisome of bacteriophage T7 assembled on DNA resembling a replication fork. A structure of the complex consisting of six domains of DNA helicase, five domains of RNA primase, two DNA polymerases, and two thioredoxin (processivity factor) molecules was determined by single-particle cryo-electron microscopy. The two molecules of DNA polymerase adopt a different spatial arrangement at the replication fork, reflecting their roles in leading- and lagging-strand synthesis. The structure, in combination with biochemical data, reveals molecular mechanisms for coordination of leading- and lagging-strand synthesis. Because mechanisms of DNA replication are highly conserved, the observations are relevant to other replication systems.

Temperature-Dependent Nanomechanics and Topography of Bacteriophage T7.

Viruses are nanoscale infectious agents which may be inactivated by heat treatment. The global molecular mechanisms of virus inactivation and the thermally induced structural changes in viruses are not fully understood. In this study, we measured the heat-induced changes in the properties of T7 bacteriophage particles exposed to a two-stage (65°C and 80°C) thermal effect, by using atomic force microscopy (AFM)-based nanomechanical and topographical measurements. We found that exposure to 65°C led to the release of genomic DNA and to the loss of the capsid tail; hence, the T7 particles became destabilized. Further heating to 80°C surprisingly led to an increase in mechanical stability, due likely to partial denaturation of the capsomeric proteins kept within the global capsid arrangement.IMPORTANCE Even though the loss of DNA, caused by heat treatment, destabilizes the T7 phage, its capsid is remarkably able to withstand high temperatures with a more or less intact global topographical structure. Thus, partial denaturation within the global structural constraints of the viral capsid may have a stabilizing effect. Understanding the structural design of viruses may help in constructing artificial nanocapsules for the packaging and delivery of materials under harsh environmental conditions.

Engineering T7 bacteriophage as a potential DNA vaccine targeting delivery vector.

BACKGROUND: DNA delivery with bacteriophage by surface-displayed mammalian cell penetrating peptides has been reported. Although, various phages have been used to facilitate DNA transfer by surface displaying the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide), no similar study has been conducted using T7 phage. METHODS: In this study, we engineeredT7 phage as a DNA targeting delivery vector to facilitate cellular internalization. We constructed recombinant T7 phages that displayed Tat peptide on their surface and carried eukaryotic expression box (EEB) as a part of their genomes (T7-EEB-Tat). RESULTS: We demonstrated that T7 phage harboring foreign gene insertion had packaged into infective progeny phage particles. Moreover, when mammalian cells that were briefly exposed to T7-EEB-Tat, expressed a significant higher level of the marker gene with the control cells infected with the wide type phage without displaying Tat peptides. CONCLUSION: These data suggested that the potential of T7 phage as an effective delivery vector for DNA vaccine transfer.

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions.

Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.

Conformational changes leading to T7 DNA delivery upon interaction with the bacterial receptor.

The majority of bacteriophages protect their genetic material by packaging the nucleic acid in concentric layers to an almost crystalline concentration inside protein shells (capsid). This highly condensed genome also has to be efficiently injected into the host bacterium in a process named ejection. Most phages use a specialized complex (often a tail) to deliver the genome without disrupting cell integrity. Bacteriophage T7 belongs to the Podoviridae family and has a short, non-contractile tail formed by a tubular structure surrounded by fibers. Here we characterize the kinetics and structure of bacteriophage T7 DNA delivery process. We show that T7 recognizes lipopolysaccharides (LPS) from Escherichia coli rough strains through the fibers. Rough LPS acts as the main phage receptor and drives DNA ejection in vitro. The structural characterization of the phage tail after ejection using cryo-electron microscopy (cryo-EM) and single particle reconstruction methods revealed the major conformational changes needed for DNA delivery at low resolution. Interaction with the receptor causes fiber tilting and opening of the internal tail channel by untwisting the nozzle domain, allowing release of DNA and probably of the internal head proteins.

Exploiting radiation damage to map proteins in nucleoprotein complexes: the internal structure of bacteriophage T7.

In the final stage of radiation damage in cryo-electron microscopy of proteins, bubbles of hydrogen gas are generated. Proteins embedded in DNA bubble sooner than free-standing proteins and DNA does not bubble under the same conditions. These properties make it possible to distinguish protein from DNA. Here we explored the scope of this technique ("bubblegram imaging") by applying it to bacteriophage T7, viewed as a partially defined model system. T7 has a thin-walled icosahedral capsid, 60 nm in diameter, with a barrel-shaped protein core under one of its twelve vertices (the portal vertex). The core is densely wrapped with DNA but details of their interaction and how their injection into a host bacterium is coordinated are lacking. With short (10 s) intervals between exposures of 17 electrons/Å(2) each, bubbling starts in the third exposure, with 1-4 bubbles nucleating in the core: in subsequent exposures, these bubbles grow and merge. A 3D reconstruction from fifth-exposure images depicts a bipartite cylindrical gas cloud in the core. In its portal-proximal half, the axial region is gaseous whereas in the portal-distal half, it is occupied by a 3 nm-wide dense rod. We propose that they respectively represent core protein and an end of the packaged genome, poised for injection into a host cell. Single bubbles at other sites may represent residual scaffolding protein. Thus, bubbling depends on dose rate, protein amount, and tightness of the DNA seal.

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.

Large terminase conformational change induced by connector binding in bacteriophage T7.

During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.

Structural characterization of the bacteriophage T7 tail machinery.

Most bacterial viruses need a specialized machinery, called "tail," to inject their genomes inside the bacterial cytoplasm without disrupting the cellular integrity. Bacteriophage T7 is a well characterized member of the Podoviridae family infecting Escherichia coli, and it has a short noncontractile tail that assembles sequentially on the viral head after DNA packaging. The T7 tail is a complex of around 2.7 MDa composed of at least four proteins as follows: the connector (gene product 8, gp8), the tail tubular proteins gp11 and gp12, and the fibers (gp17). Using cryo-electron microscopy and single particle image reconstruction techniques, we have determined the precise topology of the tail proteins by comparing the structure of the T7 tail extracted from viruses and a complex formed by recombinant gp8, gp11, and gp12 proteins. Furthermore, the order of assembly of the structural components within the complex was deduced from interaction assays with cloned and purified tail proteins. The existence of common folds among similar tail proteins allowed us to obtain pseudo-atomic threaded models of gp8 (connector) and gp11 (gatekeeper) proteins, which were docked into the corresponding cryo-EM volumes of the tail complex. This pseudo-atomic model of the connector-gatekeeper interaction revealed the existence of a common molecular architecture among viruses belonging to the three tailed bacteriophage families, strongly suggesting that a common molecular mechanism has been favored during evolution to coordinate the transition between DNA packaging and tail assembly.

Electrospun water-soluble polymer nanofibers for the dehydration and storage of sensitive reagents.

The ability to preserve and deliver reagents remains an obstacle for the successful deployment of self-contained diagnostic microdevices. In this study we investigated the ability of bacteriophage T7 to be encapsulated and preserved in water soluble nanofibers. The bacteriophage T7 was added to mixtures of polyvinylpyrrolidone and water and electrospun onto a grounded plate. Trehalose and magnesium salts were added to the mixtures to determine their effect on the infectivity of the bacteriophage following electrospinning and during storage. The loss of T7 infectivity was determined immediately following electrospinning and during storage using agar overlay plating and plaque counting. The results indicate that the addition of magnesium salts protects the bacteriophage during the relatively violent and high voltage electrospinning process, but is not as effective as a protectant during storage of the dried T7. Conversely, the addition of trehalose into the electrospinning mix has little effect on the electrospinning, but a more significant role as a protectant during storage.

Nanotip analysis for dielectrophoretic concentration of nanosized viral particles.

Rapid and sensitive detection of low-abundance viral particles is strongly demanded in health care, environmental control, military defense, and homeland security. Current detection methods, however, lack either assay speed or sensitivity, mainly due to the nanosized viral particles. In this paper, we compare a dendritic, multi-terminal nanotip ('dendritic nanotip') with a single terminal nanotip ('single nanotip') for dielectrophoretic (DEP) concentration of viral particles. The numerical computation studies the concentration efficiency of viral particles ranging from 25 to 100 nm in radius for both nanotips. With DEP and Brownian motion considered, when the particle radius decreases by two times, the concentration time for both nanotips increases by 4-5 times. In the computational study, a dendritic nanotip shows about 1.5 times faster concentration than a single nanotip for the viral particles because the dendritic structure increases the DEP-effective area to overcome the Brownian motion. For the qualitative support of the numerical results, the comparison experiment of a dendritic nanotip and a single nanotip is conducted. Under 1 min of concentration time, a dendritic nanotip shows a higher sensitivity than a single nanotip. When the concentration time is 5 min, the sensitivity of a dendritic nanotip for T7 phage is 10(4) particles ml(-1). The dendritic nanotip-based concentrator has the potential for rapid identification of viral particles.

A protein linkage map of Escherichia coli bacteriophage T7.

Genome sequencing projects are predicting large numbers of novel proteins, whose interactions with other proteins must mediate the function of cellular processes. To analyse these networks, we used the yeast two-hybrid system on a genome-wide scale to identify 25 interactions among the proteins of Escherichia coli bacteriophage T7. Among these is a set of six interactions connecting proteins that function in DNA replication and DNA packaging. Remarkably, two genes, arranged such that one entirely overlaps the other and uses a different reading frame, encode interacting proteins. Several of the interactions reflect intramolecular associations of different domains of the same polypeptide, suggesting that the two-hybrid assay may be useful in the analysis of protein folding. This global approach to protein-protein interactions may be applicable to the analysis of more complex genomes whose sequences are becoming available.

[Study of elongation complexes for T7 RNA polymerase].

Complexes of bacteriophage T7 RNA polymerase with a DNA template for transcription elongation were visualized by atomic force microscopy. Images for complexes of T7 RNA polymerase with terminal fragments of DNA template were obtained for single molecules. Complexes of a single DNA template molecule with several T7 RNA polymerase molecules corresponding to stages of initiation, elongation and termination of transcription were visualized under the elimination of unspecific DNA-protein binding. Immobilized on the amino mica RNA transcripts form rod-like condensed structures. Detailes of specific and unspecific complex formation for the T7 RNA polymerase-DNA system during initiation and transcription elongation are discussed.

DNA packaging and delivery machines in tailed bacteriophages.

Several symmetric and asymmetric reconstructions of bacteriophage particles have recently been determined using electron cryo-microscopy and image reconstruction, and X-ray crystal structures of phage particles and particle-associated gene products have also been solved. In the past two years, the asymmetric structures of four different phages, T7, epsilon15, P22 and phi29, were determined at resolutions sufficient to visualize details of the machinery for DNA packaging and delivery, as well as the organization of the double-stranded DNA within the particles. Invariably, the portals, through which DNA enters and leaves the particle, have 12-fold symmetry, occupy a pentavalent site in the capsid and, along with tail machine accessory proteins attached to it, are fixed in a specific orientation relative to the rest of the capsid.

[Visualization of elongation complexes for t7 Rna polymerase by atomic force microscopy].

Complexes of bacteriophage T7 RNA polymerase (RNAP) with a DNA template for transcription elongation were visualized by atomic force microscopy (AFM). Fragment of pGEMEX linear DNA with length of 1414 bp carrying promot- er and terminator of bacteriophage T7 was DNA template for transcription. Promoter and terminator are located unsymmetrically on the ends of DNA template. Images of stable complexes of T7 RNAP with terminal fragments of DNA template were obtained for single molecules. Complexes of a single DNA template molecule with 2-3 T7 RNAP molecules corresponding to stages of initiation, elongation and termination of transcription were visualized under the elimination of non-specific DNA-protein binding. Also complexes of DNA, RNAP and RNA transcripts were imaged. Our results suggest that the initial stage is the formation of complex between T7 RNAP and terminal fragment of DNA template. Because promoter is localized near DNA terminus, it makes impossible an ommision of the promoter site.

Stepwise reversible nanomechanical buckling in a viral capsid.

Viruses are nanoscale infectious agents constructed of a proteinaceous capsid that protects the packaged genomic material. Nanoindentation experiments using atomic force microscopy have, in recent years, provided unprecedented insight into the elastic properties, structural stability and maturation-dependent mechanical changes in viruses. However, the dynamics of capsid behavior are still unresolved. Here we used high-resolution nanoindentation experiments on mature, DNA-filled T7 bacteriophage particles. The elastic regime of the nanoindentation force trace contained discrete, stepwise transitions that cause buckling of the T7 capsid with magnitudes that are integer multiples of ∼0.6 nm. Remarkably, the transitions are reversible and contribute to the rapid consolidation of the capsid structure against a force during cantilever retraction. The stepwise transitions were present even following the removal of the genomic DNA by heat treatment, indicating that they are related to the structure and dynamics of the capsomeric proteins. Dynamic force spectroscopy experiments revealed that the thermally activated consolidation step is ∼104 times faster than spontaneous buckling, suggesting that the capsid stability is under strong dynamic control. Capsid structural dynamics may play an important role in protecting the genomic material from harsh environmental impacts. The nanomechanics approach employed here may be used to investigate the structural dynamics of other viruses and nanoscale containers as well.

Structure of the connector of bacteriophage T7 at 8A resolution: structural homologies of a basic component of a DNA translocating machinery.

The three-dimensional structure of the bacteriophage T7 head-to-tail connector has been obtained at 8A resolution using cryo-electron microscopy and single-particle analysis from purified recombinant connectors. The general morphology of the T7 connector is that of a 12-folded toroidal homopolymer with a channel that runs along the longitudinal axis of the particle. The structure of the T7 connector reveals many structural similarities with the connectors from other bacteriophages. Docking of the atomic structure of the varphi29 connector into the three-dimensional reconstruction of T7 connector reveals that the narrow, distal region of the two oligomers are almost identical. This region of the varphi29 connector has been suggested to be involved in DNA translocation, and is composed of an alpha-beta-alpha-beta-beta-alpha motif. A search for alpha-helices in the same region of the T7 three-dimensional map has located three alpha-helices in approximately the same position as those of the varphi29 connector. A comparison of the predicted secondary structure of several bacteriophage connectors, including among others T7, varphi29, P22 and SPP1, reveals that, despite the lack of sequence homology, they seem to contain the same alpha-beta-alpha-beta-beta-alpha motif as that present in the varphi29 connector. These results allow us to suggest a common architecture related to a basic component of the DNA translocating machinery for several viruses.

Can Changes in Temperature or Ionic Conditions Modify the DNA Organization in the Full Bacteriophage Capsid?

We compared four bacteriophage species, T5, λ, T7, and Φ29, to explore the possibilities of DNA reorganization in the capsid where the chain is highly concentrated and confined. First, we did not detect any change in DNA organization as a function of temperature between 20 to 40 °C. Second, the presence of spermine (4+) induces a significant enlargement of the typical size of the hexagonal domains in all phages. We interpret these changes as a reorganization of DNA by slight movements of defects in the structure, triggered by a partial screening of repulsive interactions. We did not detect any signal characteristic of a long-range chiral organization of the encapsidated DNA in the presence and in the absence of spermine.