DNA poised for release in bacteriophage phi29 - PubMed (original) (raw)

DNA poised for release in bacteriophage phi29

Jinghua Tang et al. Structure. 2008 Jun.

Abstract

We present here the first asymmetric, three-dimensional reconstruction of a tailed dsDNA virus, the mature bacteriophage phi29, at subnanometer resolution. This structure reveals the rich detail of the asymmetric interactions and conformational dynamics of the phi29 protein and DNA components, and provides novel insight into the mechanics of virus assembly. For example, the dodecameric head-tail connector protein undergoes significant rearrangement upon assembly into the virion. Specific interactions occur between the tightly packed dsDNA and the proteins of the head and tail. Of particular interest and novelty, an approximately 60A diameter toroid of dsDNA was observed in the connector-lower collar cavity. The extreme deformation that occurs over a small stretch of DNA is likely a consequence of the high pressure of the packaged genome. This toroid structure may help retain the DNA inside the capsid prior to its injection into the bacterial host.

PubMed Disclaimer

Figures

Fig 1

Fig 1

Solid-surface representations of the fiberless ϕ29 virion and empty particle 3D reconstructions, segmented and color-coded to highlight different components. (A) Stereo view of the virion with a Q=5 lattice (gray) added to highlight the arrangement of pentameric and hexameric capsomeres in the prolate head. The BIG2 protrusion domains in one hexamer of gp8 are highlighted in red. (B) Monoscopic view of virion with front half removed to show interior components. (C) Same as (b) for empty particle. Color scheme: capsid (gp8) - blue; connector (gp10) – yellow; lower collar and tube (gp11) – green; knob (gp9) – cyan; appendages (gp12*) – magenta; terminal protein (gp3) on right end of DNA – white; and DNA alone - red. Density threshold (~1σ) yields the expected volume of the components.

Fig 2

Fig 2

Density sections from the tail regions in the fiberless ϕ29 full (A–C) and empty (D–F) particles. Features with highest or lowest densities appear darkest and lightest, respectively. (A) Longitudinal section of the virion reconstruction labeled to highlight the locations of some phage components. (B,C) Density sections normal to the tail axis at the locations depicted by dashed lines in (A) intersect two portions of the top of the gp10 connector in (B) and the middle of the gp11 lower collar in (C). (D–F) Same as (A–C) for the empty particle. The predominant density features, seen in the virion but not in the empty particle reconstructions, are identified as belonging to DNA and gp3. These include i) the ring of ~190Å diameter that encircles the gp10 dodecamer inside the capsid (B), ii) linear columns positioned along the phage axis and traversing the connector (A,B) and upper tail tube (A), iii) the ~60Å diameter toroid in the connector-collar cavity (A,C), and iv) more diffuse density in the lower portion of the tail tube, terminating at the entrance to the tail knob cavity (A). The twelve asymmetrically arranged copies of gp10 are labeled in (E) but not (B). Scale bar in (F) applies to all panels.

Fig 3

Fig 3

Connector structure in the fiberless ϕ29 virion and in crystals. (A) Stereo view of a portion of the connector region in the reconstructed virion density map (gray wire mesh; ~2σ threshold) into which an atomic model (magenta ribbon diagram) of the X-ray crystal structure of the connector (Guasch et al., 2002) was docked. The gp10 subunits of the model were adjusted, essentially as a set of rigid bodies, to best fit the prominent α-helices into the corresponding tubes of density in the reconstructed 3D map. (B) Tertiary structure of the gp10 monomer in crystals of the symmetric dodecamer (Guasch et al., 2002). Top and central (in red), and bottom (cyan) domains in gp10 are identified. Highly flexible regions, invisible in the gp10 X-ray structure are indicated by dashed curves. Helices (α1–α6) are labeled according to their location in the primary amino acid sequence (α6 is kinked, with short and long segments). (C) Stereo view showing a ribbon diagram of the gp10 crystal structure (red and cyan) superimposed with a fitted virion model (magenta). The bottom domain (B) was not modeled in the virion density map (see text).

Fig 4

Fig 4

DNA organization in ϕ29. (A) Cutaway view of the ϕ29 virion head, showing the compact layers in the bulk DNA (red). Four layers are clearly visible, with the outermost one closely following and interacting with the capsid (blue) inner surface in several places (e.g. arrows). (B) Near axial view of the virion, looking towards the tail with most of the capsid density removed. This highlights numerous contacts between the capsid and bulk DNA. These contacts, which are colored blue but may include protein, DNA or a mixture, predominantly occur near the same end of the long α-helix in each gp8 (green helical ribbons) and cluster near the Q=5 lattice edges (yellow lines). (C) Structure of DNA, segmented from the mature phage (Fig. 1B), viewed from below the tail towards the top of the head. The outermost layer of the bulk DNA contains numerous latitudinal striations as well as a ring of density that encircles but does not contact the connector at the base of the head. Two linear columns of density, each ~20Å in diameter, and the ~60Å diameter toroid sandwiched between them lie along the tail axis. Density thresholds set to ~1σ (panels A and B) and ~2σ (panel C). The higher threshold in (C) was chosen to emphasize the DNA packing.

Fig 5

Fig 5

DNA structure in tail of ϕ29 virion. (A) Stereo view of the reconstructed virion density map (gray wire mesh; ~2σ threshold) in the region near the tail into which a model (magenta ribbon diagram) of the gp10 structure (Guasch et al., 2002) was fitted. Two columns of density, along the tail axis, lie above and below a doughnut-shaped feature centered in the cavity formed by gp10 and gp11. The density threshold of the map was raised to help emphasize boundaries between viral components. (B) Monoscopic view at lower density threshold than used in (A) shows two segments of linear, dsDNA atomic models (space-filling representation), placed into the columns of density. These models do not represent accurate, fitted structures but they do illustrate that the density features are consistent with the size and dimensions of dsDNA.

Fig 6

Fig 6

DNA path through the connector, lower collar, and tail tube viewed in segmented representations of the ϕ29 virion density map (~2σ threshold). (A) A cylindrical column of high density emerges from the bulk DNA (red) in the head, slightly above the connector (yellow), and follows a path along the connector-tail axis into the gp10/11 cavity where it transforms into a toroid-shaped structure. From there, a column of high density emerges at the bottom of the toroid, continues for ~80 Å along the tail tube axis, after which weaker density (white) fills the rest of the tube, ending at the gp9 tail knob cavity (not shown). Four arrows identify regions in the plane of the cross section where the DNA and gp10 (yellow) and gp11 (green) are in close contact. These contacts, along with the apparent tight interactions between the DNA-gp3 and the end of the tail tube, might collectively help prime the highly pressurized DNA for release upon attachment of the phage to the host. (B) Tilted, close-up view of the DNA toroid and its surroundings. Color scheme matches that used in Figure 1.

Comment in

Similar articles

Cited by

References

    1. Aebi U, Bijlenga R, v d Broek J, v d Broek H, Eiserling F, Kellenberger C, Kellenberger E, Mesyanzhinov V, Muller L, Showe M, et al. The transformation of tau particles into T4 heads. II. Transformations of the surface lattice and related observations on form determination. J. Supramol. Struct. 1974;2:253–275. - PubMed
    1. Agirrezabala X, Martín-Benito J, Castón JR, Miranda R, Valpuesta JM, Carrascosa JL. Maturation of phage T7 involves structural modification of both shell and inner core components. Embo J. 2005;24:3820–3829. - PMC - PubMed
    1. Allen GS, Frank J. Structural insights on the translation initiation complex: ghosts of a universal initiation complex. Mol. Microbiol. 2007;63:941–950. - PubMed
    1. Anderson D, Reilly B. Bacteriophage □29 morphogenesis. In: Hoch JA, Losick R, Sonenshein AL, editors. Bacillus subtilis and Other Gram Positive Bacteria: Physiology, Biochemistry, and Molecular Genetics. Washington, D.C.: ASM Publications; 1993. pp. 859–867.
    1. Baker ML, Jiang W, Rixon FJ, Chiu W. Common ancestry of herpesviruses and tailed DNA bacteriophages. J. Virol. 2005;79:14967–14970. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources