Structure and assembly of the essential RNA ring component of a viral DNA packaging motor

Abstract

Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage 29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 Å resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of 29 DNA.

Fig. 1.

The pRNA is an essential component of the bacteriophage ϕ29 DNA packaging motor. (A) Model of the ϕ29 DNA packaging motor derived from cryo-EM 3D reconstructions (3). The head-tail connector (green), pRNA (magenta), and gp16 ring-ATPase (blue) are positioned at the portal vertex of the prohead. (B) Sequence and secondary-structure model of the 120b pRNA Domain I. Nucleotides included in the crystal structure are colored: gray, P_A; green, three-way junction; violet, P_C; orange, L_CE; magenta, P_E and L_E; and cyan, P_D and L_D. The crystallized pRNA oligomerization domain is in the boxed area. In light gray are nucleotides deleted in the final crystallization construct (pRNAmin). The 4-bp P_A sequence was changed to a 5′-GGCG helix (12). Residue numbers refer to the wild-type sequence. (C) The I422 crystal lattice of pRNAmin viewed along the fourfold symmetry axis, revealing the tetrameric pRNA ring. The 3D lattice is formed via P_A–P_A and P_E–P_E crystal stacking above and below.

Fig. 2.

Crystal structure of the tetrameric pRNA ring. (A) Side and top-down views of the tetrameric pRNA ring assembled via protomer head-to-tail base-pairing interactions. (B) Stereo view of the pRNA protomer, which adopts an extended conformation resembling the letter “τ” rotated counterclockwise. (C) Stereo view of the intermolecular interface. The L_CE–L_D interaction mediates the continuous base stacking from P_E of one pRNA protomer to P_A of its neighbor, creating an RNA superhelix. All coloring schemes are as in panel B.

Fig. 3.

Docking of the pRNA crystal structure into the ϕ29 prohead cryo-EM envelopes. (A) Side and bottom views of the pentameric pRNA ring structure flexibly docked into its cryo-EM envelope (21). (B) Side and bottom views of the pentameric pRNA ring and the dodecameric connector structures (2) fit into their corresponding cryo-EM envelopes. Note the gap between the pRNA and the connector at the L_E region. (C) Side view of the pRNA ring and a pseudoatomic model of the gp8 capsid protein fit into the cryo-EM envelope. Note the pRNA-gp8 contacts in the L_E region. The connector structure was omitted for clarity. (D) Side view of the modeled 120b pRNA pentameric ring superimposed with the cryo-EM envelopes of the pRNA and the pRNA-bound gp16 ATPase (3), contoured at 1.7σ. The ATPase density is weakened at this contour level. Note the inferred pRNA–gp16 interactions at the lower portion of P_A. (E, F) Effect of L_E loop substitutions on in vitro DNA packaging and prohead binding. (E) Proheads reconstituted with 120b pRNA having either the L_E-to-GAAA or -UUCG tetraloop substitution were tested for in vitro DNA packaging. The packaged DNA protected from DNase digestion was extracted from the phage head and analyzed on an agarose gel. The Input lane shows the quantity of DNA added to a packaging reaction. The negative control omits ATP from the packaging reaction. (F) For prohead binding, proheads were incubated with pRNAs with L_E loop substitutions, purified, and RNA content analyzed on denaturing urea-PAGE.

Fig. 4.

SHAPE chemical probing and footprinting analysis of pRNA conformation and its interactions with the ϕ29 prohead. (A) SHAPE reactivity of the free and prohead-bound pRNA. The residue numbers and color-coded secondary structures are marked beside the SHAPE lanes. The adenosine sequencing ladder (lane 1) migrates 1-nt faster than the SHAPE lanes. Background due to reverse transcriptase pausing is shown in lane 4. (B) Quantitative analysis of the pRNA SHAPE reactivity profile to reveal “footprints” due to pRNA-prohead interactions. Positive values indicate protection due to prohead binding. (C) Protected regions of pRNA (in magenta) were mapped onto the ϕ29 prohead-pRNA structure model.

Fig. 5.

Activity of designer pRNAs. (A) A designer pRNA or variants where the J_u flexible bulge was replaced with adenines or with the L_E-to-UUCG substitution were generated. These RNAs were then assayed for in vitro DNA packaging activity (B) and prohead binding (C).