Biochemical studies on DNA -packaging RNA essential for bacteriophage phi29 genome encapsidation

Bacteriophage phi29 packages its double stranded genomic DNA-gp3 into a preformed procapsid during replication. A highly sensitive completely in vitro assembly system of phi29 has been established, providing a simple system to study dsDNA packaging. One of most fascinating characteristics of phage phi29 is that a virally encoded 120 base long pRNA is absolutely required for the DNA packaging process. This pRNA bound to procapsids in the presence of Mg++ and left the DNA-filled head after packaging process is finished. A stoichiometry study showed that six pRNAs bind to one procapsid. Competition and inhibition assay suggested that six pRNAs worked sequentially to rotate the DNA packaging machinery to screw genomic DNA-gp3 into procapsids, similar to a bolt through a hexa nut. In order to turn the motor there must be a tremendous accompanying conformational change accompanied with. Indeed, Mg-induced conformational changes of pRNA were identified using ribonuclease probing, psoralen and rhodium (iii) complex crosslinking. The finding of inter-pRNA interaction further supported the sequential model of pRNAs. Base pairing between two loops of pRNA intermolecularly was important for inter-pRNA interaction. The pRNAs with inter-locking loop sequences can form dimers and trimers in solution. Stable pRNA dimers and trimers were isolated from native gels, separated by sucrose gradient ultracentrifugation and observed by cryo-AFM, providing a model to study RNA/RNA interaction in a protein-free solution. A 75 nucleotides RNA segment, bases 23–97, was able to form dimers, to interlock into hexamers, to compete with full length pRNA for procapsid binding, and therefore to inhibit phi29 assembly in vitro. Our result suggested that segment 23–97 was a self-folded independent domain involved in procapsid binding and RNA/RNA interaction in dimer and hexamer formation, while bases 1–22 and 98–120 are involved in DNA translocation but dispensable for RNA/RNA interaction. The pRNA dimer was the building block in hexamer formation, and the pathway of forming a hexamer is: dimer → tetramer → hexamer. Interaction between two adjacent loops played a key role in recruiting the in-coming dimer. Procapsid served as the foundation for dimer binding to assemble hexamer and formation of a functional hexamer required specific divalent ions