Biophysical and functional studies of pRNA: A self-associating RNA

The packaging RNA (pRNA) found in the phi29 family of bacteriophage is an essential component of a powerful molecular motor used to package the phage's DNA genome into the capsid. The pRNA forms a higher-order multimer by intermolecular "kissing" interactions between identical molecules and is the only known naturally-occurring self-associating RNA. pRNAs could be building blocks for nanotechnology and they are good models to explore the principles of RNA self-association. To uncover rules that could guide rational engineering of pRNA self-association, I examined the multimerization behavior of pRNAs from different phi29 family members, pRNA mutants, and chimeric pRNAs using a combination of biochemical and biophysical methods. I discovered that phylogenetically related pRNAs from phi29 family phages have diverse self-association properties. I found that the three-way junction (3wj) within each pRNA, despite not making direct intermolecular contacts, plays important roles in stabilizing the intermolecular interactions and dictating the size of the multimer formed (dimer, trimer, etc.). Specifically, the 3wj in the pRNA from phage M2 appears to favor a different tertiary conformation compared to the 3wj in the pRNA from phi29, and M2's junction facilitates formation of a higher order multimer that is more thermostable than phi29's. This previously undescribed behavior may provide us new scaffolds to engineer fine-tuned behaviors into pRNAs and also shed light on the mechanisms of RNA self-association. Ongoing efforts include developing a system to explore pRNA's role in phage and solving a high-resolution pRNA structure.