A three-helix junction is the interface between two functional domains of prohead RNA in 29 DNA packaging

Abstract

The double-stranded-DNA bacteriophages employ powerful molecular motors to translocate genomic DNA into preformed capsids during the packaging step in phage assembly. Bacillus subtilis bacteriophage 29 has an oligomeric prohead RNA (pRNA) that is an essential component of its packaging motor. The crystal structure of the pRNA-prohead binding domain suggested that a three-helix junction constitutes both a flexible region and part of a rigid RNA superhelix. Here we define the functional role of the three-helix junction in motor assembly and DNA packaging. Deletion mutagenesis showed that a U-rich region comprising two sides of the junction plays a role in the stable assembly of pRNA to the prohead. The retention of at least two bulged residues in this region was essential for pRNA binding and thereby subsequent DNA packaging. Additional deletions resulted in the loss of the ability of pRNA to multimerize in solution, consistent with the hypothesis that this region provides the flexibility required for pRNA oligomerization and prohead binding. The third side of the junction is part of a large RNA superhelix that spans the motor. The insertion of bases into this feature resulted in a loss of DNA packaging and an impairment of initiation complex assembly. Additionally, cryo-electron microscopy (cryoEM) analysis of third-side insertion mutants showed an increased flexibility of the helix that binds the ATPase, suggesting that the rigidity of the RNA superhelix is necessary for efficient motor assembly and function. These results highlight the critical role of the three-way junction in bridging the prohead binding and ATPase assembly functions of pRNA.

Fig 1

The DNA packaging motor of bacteriophage ϕ29. (a) CryoEM reconstruction of the ϕ29 prohead (left) and with the packaging motor complex (right, cutaway), showing the molecular envelopes of the motor components. The head/tail connector is in green, the pentameric pRNA is in pink, and the packaging ATPase gp16 is in blue. A DNA molecule has been placed into the channel for reference. (Reproduced from reference with permission from Elsevier.) (b) Predicted secondary structure of the 120-base pRNA (2). The helices and loops of pRNA are denoted by letters. Bases 25 to 95 comprise the prohead binding domain that was solved by X-ray crystallography (9). The shaded region represents the footprint of the prohead on bound pRNA (30). Oligomerization of pRNA occurs via intermolecular base pairing between the CE- and D-loops of adjacent pRNA molecules (18, 37). The 3-way helix junction is comprised of the single-base bulge U29, the tri-U linker (bases 72 to 74), and the intersection where the A- and D-helices meet (between bases U91 and C92). (c, left) Close-up of the crystal structure (PDB accession number 3R4F) (9) of the three-helix junction in a pRNA monomer, with bases U72 to U74 in blue, U29 in red, and the A-D junction in magenta. (Right) Model of the crystal structure of the pRNA-prohead binding domain flexibly docked into the pentameric cryoEM map of the pRNA density (9, 26).

Fig 2

Analysis of the U-rich region (U29 and U72-74) of the three-helix junction of pRNA. (a) In vitro DNA packaging by proheads containing pRNA with deletions in the U-rich junction. To assess packaging efficiency, the packaged DNA that was protected from DNase treatment was extracted from particles and analyzed by agarose gel electrophoresis. Packaging was assessed by using proheads reconstituted with wild-type (wt) pRNA (lane 2), pRNA with deletions in the tri-U linker (bases 72 to 74) (lanes 3 to 5), a deletion of the single-base U29 bulge (lane 6), or deletions in both regions (lanes 7 and 8). Input represents DNA added to the packaging reaction mixture (lane 1). In the negative control, ATP was omitted from the packaging reaction mixture (lane 9). (b) Prohead binding by deletion mutants in the U-rich region. RNA-free proheads were mixed with wild-type or mutant pRNA, and the prohead-pRNA complexes were purified away from unbound pRNA. The RNA contents of these particles were analyzed on a denaturing urea-PAGE gel. Shown are the RNA contents of proheads with wild-type pRNA (lanes 1 and 4), pRNA with deletions in the tri-U linker (bases 72 to 74) (lanes 2 and 3), and pRNA with deletions of U29 and tri-U-linker bases (lanes 5 and 6). The 120-base pRNA is a standard to mark the position of pRNA (lane 7). (c) Multimerization of pRNA deletion mutants. Free pRNA was incubated in the presence of Mg²⁺, and its oligomeric state was assessed on a native-PAGE gel. Wild-type pRNA (lane 1) and deletions in the tri-U linker (lanes 2 to 4) were analyzed. pRNA mutant F6, which has mutated residues that prevent multimerization, was used as a control to mark the position of monomeric pRNA (lane 5) (37).

Fig 3

Analysis of the third side of the three-helix junction. (a) Model of the pentameric 120-base pRNA, created from the crystal structure of the prohead binding domain with a modeled A-helix, fit into the cryoEM map of the prohead-pRNA-ATPase motor complex (9). The pRNA superhelical scaffold would be extensive, connecting the shell, connector, and ATPase. (Reproduced from reference with permission of the publisher.) (b) In vitro DNA packaging by proheads containing pRNA insertion mutants. Packaging was assessed by using proheads reconstituted with wild-type pRNA (lane 2) or U insertions (ins.) between bases 91 and 92 (lanes 3 and 4). (c) Prohead binding by insertion mutants of pRNA. Proheads were reconstituted with wild-type pRNA (lane 1) or U insertion mutant pRNA (lanes 2 and 3) and purified away from unbound pRNA, and the RNA content was analyzed by denaturing urea-PAGE. The 120-base pRNA is a standard to mark the position of pRNA (lane 4).

Fig 4

Motor function of insertion mutants. (a) Ternary complex formation by insertion mutants. Proheads with wild-type or mutant pRNAs were incubated with the packaging ATPase gp16 and [³H]DNA-gp3, and the complexes were isolated by use of magnetic beads coated with anti-ϕ29 antibodies. Error bars represent standard deviations (n = 3). (b) ATPase activity during DNA packaging. Packaging complexes with wild-type or insertion mutant pRNAs were assembled, and P_i production due to ATPase activity was monitored via a colorimetric change detected at an A₃₆₀. P + gp16 is a DNA-free mixture containing proheads with U92 insertion pRNA and gp16, representing ATPase activity not associated with DNA packaging. gp16 alone lacks proheads, and DNA-gp3 and represents the basal ATPase activity. Shown is a trace representative of data from 3 experiments. OD 360, optical density at 360 nm.

Fig 5

CryoEM 3D reconstruction of pRNA insertion mutants on the third side of the three-helix junction. Proheads contained (a) wild-type pRNA (26); (b) pRNA with a deletion of the essential CCA bulge demonstrates the detection of an altered A-helix of pRNA (39); (c) U92 insertion pRNA; and (d) U92-93 insertion pRNA. CryoEM maps of proheads with wild-type pRNA and CCA deletion pRNA (a and b, respectively) were low-pass filtered to 18 Å for comparison with the reconstructions of the U92 and U92-93 insertion mutants.