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.
Highlights
Motor can package the 19,300 bases of the 29 DNA genome into the viral capsid against a load as high as 57 piconewtons with a maximum force exceeding 100 piconewtons, filling the capsid to Ͼ50% capacity and reaching internal pressures as high as 60 atm (16 –19)
Cryo-electron microscopy of in vitro assembled packaging motors showed a ring of five Prohead RNA (pRNA) interacting with a ring of five gp16 ATPases and a ring of 12 gp10 connector proteins to form the core of the nanomotor [12]
PRNA Energetics and Self-assembly side the interlocking loops may compensate for the different stabilities of loop-loop interactions
Summary
RNA Preparation—The sequences of M2 and GA1 pRNAs were cloned from genomic DNA provided by Dr Shelley Grimes (University of Minnesota). Sedimentation Velocity Analysis—Analytical ultracentrifugation experiments were conducted in the same buffer conditions as used for native gels to determine the molecular weight of gel bands. The rotor and cells were pre-equilibrated at 4 °C, and the samples were renatured from 90 to 4 °C by snap cooling on ice. The buffer for the sample was 90 mM Tris, 200 mM boric acid, 5 mM NaCl, and 10 mM MgCl2 (pH 7.5). After at least 30 min of equilibration, the pRNA sample was mixed with 40% (w/v) sucrose loading buffer and run on a 10% polyacrylamide gel buffered at pH 7.5 in 90 mM Tris and 200 mM boric acid with different concentrations of NaCl or MgCl2. The pRNA samples are 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 molecular weights of species 2 are consistent with a dimer conformation. —, no conformation was observed with this molecular weight
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