2008 Rustbelt RNA Meeting
Linear dsDNA viruses, including bacteriophage phi29, translocate their genome into a pre-assembled procapsid to near-crystalline density. DNA packaging motor of phi29 uses dodecameric connector as a DNA entry channel, hexameric packaging RNA (pRNA) and packaging enzyme gp16 to drive force to overcome the unfavorable packaging reaction.
Biochemical studies showed that 5’/3’ paired helical region of pRNA in the pRNA/procapsid complex is for gp16 binding, while its central domain binds to the procapsid. pRNA bulge C18C19A20 that is essential for DNA packaging was dispensable for gp16 binding. The interaction of gp16 with nucleic acids was also investigated using recently developed single molecule TIRF system. The study showed that gp16 binds nucleic acids (RNA or DNA) depending on their structures (single-stranded or double-stranded) and chemistry (purine or pyrimidine). Gp16 contains a conserved nucleotide triphosphate binding motif. The steady-state analysis showed that pRNA or DNA binding stimulates the ATP hydrolysis of gp16. The stimulation extent was dependent on the structure and chemistry of DNA or RNA, strongly suggesting that gp16 interacts with pRNA as a part of ATP hydrolyzing complex in the packaging motor, and its ATPase activity can be stimulated via their interactions. To understand the mechanism of the motor action, the kinetics of the ATPase activity of gp16 was determined as a function of DNA structure or chemistry. The kcat and Km in the absence of DNA was 0.016/s and 351.0 µM, respectively, suggesting that gp16 itself is a slow-ATPase with a low affinity for substrate. The affinity of gp16 for ATP was greatly boosted by the presence of pRNA or DNA, but the ATPase rate was strongly affected by DNA structure and chemistry. The order of ATPase stimulation is poly d(pyrimidine) > dsDNA > poly d(purine), which agreed with the order of the DNA binding to gp16, as revealed by single-molecule fluorescence microscopy. Interestingly, the stimulation degree by phi29 pRNA was similar to that of poly d(pyrimidine). The results suggest that pRNA accelerates gp16 ATPase activity more significantly than genomic dsDNA, albeit both pRNA and genomic DNA are involving in contact with gp16 during DNA packaging.
Keywords: phi29 DNA packaging motor, pRNA, packaging enzyme