Constructing an active chimeric pRNA ring with a stoichiometry of six and identifying 12 domains of the pRNA ring binding to the 12-subunit channel of phi29 DNA-packaging motor.

During the last stage of replication of double-stranded RNA or DNA viruses, their genome is packaged into a preassembled protein capsid. The bacterial virus phi29 dsDNA-packaging motor uses a noncoding packaging RNA (pRNA) molecule to gear its genomic DNA translocation. In this study, we constructed chimeric pRNAs by fusing the pRNA of bacterial virus M2 and that of phi29. The chimeric pRNAs can form dimers or trimers. The dimeric or trimeric pRNAs were active in the packaging of the phi29 dsDNA genome into the purified procapsid, which was subsequently converted into the infectious viruses, as proven by counting plaque-forming units (PFUs). These data show that the stoichiometry of the chimeric pRNAs on the motor is six subunits, a multiple of 2 and 3. Furthermore, AFM studies on pRNA fused to an RNA-triangle revealed hexamer formation. But how do the six identical RNAs anchor on the 12-subunit connector with the double stoichiometry? Structural analysis in combination with enzymatic and chemical probing data revealed that each native pRNA contributes two domains to bind to the 12-subunit DNA-packaging channel at three positively charged residues RKR, proving the formation of the hexameric ring. Resolving the hexamer versus pentamer debate clarifies the mechanism of dsDNA translocation in living organisms.