Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages

Abstract

Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a "small terminase" and a "large terminase" component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.

Fig. 1.

Ribbon diagrams of the small terminase of phage 44RR. (A) Ribbon diagram of a monomer from the 12-mer assembly. The termini are labeled N and C, and selected amino acids are numbered. (B and C) Stereo diagrams of the 12-mer assembly in views related by 90° rotation. The ribbon diagrams were generated with the Chimera program (51).

Fig. 2.

Structural comparisons with other small terminases. (A) Superposition of 44RR gp16 (cyan) with Sf6 gp1 (yellow) and SF6 G1P (magenta). (B) Ribbon diagrams of two neighboring monomers from three small terminases showing that the relationship between the monomers are different in 44RR and SF6 compared to Sf6. In 44RR and SF6, helix α₂ packs against a crevice formed by α₁ and α₂ in the neighboring counterclockwise rotated monomer when viewed from the crown, whereas in Sf6, α₂ packs against a crevice formed by α₁ and α₂ in the neighboring clockwise rotated monomer.

Fig. 3.

Biochemical characterizations of gp16 domain constructs. (A) Schematic of the gp16 domain constructs. Numbers represent the number of amino acids in the gp16 coding sequence. The gp16 domain construct polypeptides are shown as horizontal bars with different colors representing different gp16 domain sequences. (B) Stimulation of gp17-ATPase. The 0.5 μM gp17 and 5 μM gp16 constructs were used for the ATPase assays. Each construct was assayed in duplicate, as indicated by the bracket. (C) Inhibition of gp17-nuclease. Various gp16 constructs were incubated with 100 ng of phage λ DNA (48.5 kb) and gp17 at the concentrations indicated. (D) Modulation of DNA packaging. Increasing concentrations of gp16 constructs were added into a reaction mixture containing 600 ng of phage λ DNA, 10¹⁰ T4 head particles, 2 μM gp17, and 1 mM ATP to investigate their modulation of DNA packaging. Levels of stimulation were calculated by dividing the elevated DNA packaging amount with the amount of DNA that was packaged without addition of any gp16 constructs. Levels of inhibition were calculated by dividing the amount of DNA packaged in the absence of any gp16 constructs with decreased amount of DNA that is packaged when gp16 constructs were present. Note that the N-del (▪) and C-del (□) symbols at 3 and 12 μM are not completely visible because the WT symbol (♦) which has the same level of inhibition masks the N-del and C-del symbols. Details on ATPase, nuclease, and DNA-packaging assays are described in SI Materials and Methods. (E) Table summarizing the characterizations of the gp16 domain constructs. Rough estimates of the nuclease inhibition activities are represented by “+” and “-” with more + representing more activity and - indicating no detectable activity. The 11-mer and 12-mer oligomeric states of gp16 constructs were determined by crystallographic data, and the dimer state was determined by gel filtration. Stimulation of DNA packaging activity is shown by “↑” and inhibition shown by “↓”.

Fig. 4.

A model for packaging initiation by the small terminase. Framed area: The small terminase subunits are represented in rainbow colors. The N-terminal ATPase domain of the large terminase is colored purple and the C-terminal nuclease domain cyan. (A) Packaging initiation sites on the phage genomic DNA. (B) Two small terminase oligomers bound to the initiation sites. (C) Two large terminase molecules are recruited and cleave the DNA backbones associated with the same base pair. The nuclease domain of the large terminase on the left-hand side is obscured by the twofold related nuclease domain of the large terminase on the right-hand side. (D) Two free ends are generated on the DNA, and further digestion is prevented by the small terminase. (E) The initiation complex binds to an empty procapsid. (F) Four more large terminase molecules are recruited to assemble the pentameric motor and rapid DNA translocation causes the dissociation of the small terminase.