Different sequences show similar quaternary interaction stabilities in prohead viral RNA self-assembly

Abstract

Prohead RNA (pRNA) is an essential component of the self-assembling ϕ29 bacteriophage DNA packaging motor. Different related species of bacteriophage share only 12% similarity in pRNA sequences. The secondary structure for pRNA is conserved, however. In this study, we present evidence for self-assembly in different pRNA sequences and new measurements of the energetics for the quaternary interactions in pRNA dimers and trimers. The energetics for self-assembly in different pRNA sequences are similar despite very different sequences in the loop-loop interactions. The architecture surrounding the interlocking loops contributes to the stability of the pRNA quaternary interactions, and sequence variation outside the interlocking loops may counterbalance the changes in the loop sequences. Thus, the evolutionary divergence of pRNA sequences maintains not only conservation of function and secondary structure but also stabilities of quaternary interactions. The self-assembly of pRNA can be fine-tuned with variations in magnesium chloride, sodium chloride, temperature, and concentration. The ability to control pRNA self-assembly holds promise for the development of nanoparticle therapeutic applications for this biological molecule. The pRNA system is well suited for future studies to further understand the energetics of RNA tertiary and quaternary interactions, which can provide insight into larger biological assemblies such as viruses and biomolecular motors.

FIGURE 1.

Secondary structures of φ29, M2, SF5, and GA1 pRNAs. Conserved nucleotides are green. Nucleotides that participate in potential Watson-Crick intermolecular tertiary and quaternary interactions between the CE bulge loop and the D loop hairpin are in red. The secondary structures shown were determined experimentally by enzymatic and chemical modification data (3). Three additional pRNA sequences have also been proposed from phylogenetic alignments (9).

FIGURE 2.

Ball-and-stick model of pRNA monomer, dimer, and pentamer. Loops are represented as balls, and helices are represented as sticks. The angles between helices are shown as right angles for simplicity, but coaxial stacking and helix orientations have not been experimentally determined yet. The helices are labeled in the monomer diagram. Helix B extends from the 3′-end of helix A in the full-length 174-mer pRNA but is not necessary for self-assembly or motor function and is not shown. The CE bulge loop and the D hairpin loop are shown as orange balls. Base pairing interactions between the CE and D loops is proposed to occur in pRNA dimers and multimers. The arrangement of pRNA in a pentamer is based on cryo-electron microscopy of in vitro assembled packaging motors (12).

FIGURE 3.

M2, GA1, SF5, and φ29 pRNA self-assembly in serial dilutions. A, the pRNA concentrations in lanes i–xiii are 0.007, 0.009, 0.01, 0.02, 0.03, 0.05, 0.1, 0.2, 0.3, 0.6, 1.3, 2.5, and 5 μm, respectively. The pRNA samples were renatured from 90 to 4 °C by snap cooling on ice in 5 mm NaCl and 10 mm MgCl₂ with 90 mm Tris and 200 mm boric acid (pH 7.5). The native polyacrylamide gel was run at 4 °C with the same buffer conditions as the pRNA sample. B, the φ29 pRNA samples were renatured from 90 to 4 °C by snap cooling on ice in 10 mm MgCl₂ with 90 mm Tris and 200 mm boric acid (pH 7.5). The pRNA concentrations in lanes i–xiii are 0.012, 0.022, 0.032, 0.051, 0.090, 0.17, 0.33, 0.64, 1.3, 2.5, 5.0, 10, and 20 μm, respectively. The gels were analyzed with ImageQuant software, and background correction values were <10%. Additional bands in the same lanes outside of the region of gel shown were not observed. The positions of monomer and dimer bands were benchmarked using analytical ultracentrifugation experiments. DNA size markers were used only as a reference for consistency in comparing many different gels (supplemental Table S3). Additional gel data at different pRNA concentrations and ionic conditions are included in supplemental Figs. S2–S6.

FIGURE 4.

Native gel analysis of pRNA mutants. The pRNA samples were renatured from 90 to 4 °C by snap cooling on ice in 5 mm NaCl and 10 mm MgCl₂ with 90 mm Tris and 200 mm boric acid (pH 7.5), but there was no MgCl₂ in the RNA samples or gels in the right panel in B. A, 20 μm mutant 1 pRNA formed only a monomer. B, 0.1 μm mutants 2–5 formed dimers in the presence of MgCl₂ but formed only monomers in the absence of MgCl₂. C, concentration dependence of dimer formation for mutants 4 and 7. The pRNA concentrations in lanes i–iv are 0.1, 0.3, 1.1, and 5.1 μm, respectively. D, concentration dependence of dimer formation for mutants 8–10. The pRNA concentrations in lanes i–iv are 0.1, 2.6, 7.6, and 20.1 μm, respectively. E, concentration dependence of dimer formation for mutants 11–13. The pRNA concentrations in lanes i–iv are 0.1, 0.3, 1.1, and 5.1 μm, respectively. F, concentration dependence of dimer formation for mutants 14 and 15. The pRNA concentrations for mutant 14 in lanes i–iv are 0.1, 0.2, 0.3, and 0.7 μm, respectively, and those for mutant 15 are 0.1, 0.3, 1.1, and 5.1 μm, respectively. The same amount of labeled ³²P was added to all samples, and the amount of monomer and dimer was calculated as a ratio of the measured intensities in monomer and dimer bands.

FIGURE 5.

Optical melting curves of φ29 (A) and GA1 (B) pRNAs at 3 μm RNA and 100 mm NaCl (yellow); 10 μm RNA and 100 mm NaCl (pink); 3 μm RNA, 100 mm NaCl, and 5 mm MgCl₂ (blue); and 10 μm RNA, 100 mm NaCl, and 5 mm MgCl₂ (purple). Abs, absorbance.