Bacterial virus phi29 translocates its genomic DNA into a pre-self-assembled protein shell, known as a procapsid, to near-crystalline density. Due to the limitation of the spatial capacity of procapsids, phi29 developed a unique ATP-driven viral DNA packaging motor to complete such an entropically unfavorable DNA packaging process. The phi29 packaging motor obtains mechanical energy via ATP hydrolysis by packaging enzyme gp16, and another key component, packaging RNA (pRNA), binds to the N-terminus of the connector that is a portal channel of procapsid for DNA entry. However, the molecular mechanism of how these components interact with each other and with DNA substrate is still unclear.This study revealed that gp16 binds DNA nonspecifically, and to the 5’/3’ paired helical region of pRNA bound to procapsid connector. It suggests that gp16 serves as a linkage between pRNA and DNA, and as an essential DNA-contacting component during DNA translocation. Further kinetic studies on the ATPase activity of gp16 showed that the presence of DNA or pRNA greatly boosted the catalytic activity and the affinity of gp16 for ATP. The stimulation order of ATPase activity 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, pRNA showed also stimulatory effect on the ATPase activity of gp16 with a similar extent of poly d(pyrimidine). The results suggest that gp16 itself is a slow-ATPase with a low affinity for substrate, and that interaction with both pRNA and genomic DNA affects the enzymatic function of gp16 during DNA packaging.
For nanotechnological application of gp16, the gene of gp16 was genetically engineered to carry the fluorescent fusion protein (eGFP). The resulting eGFP-gp16 was shown to be biologically active in both ATP hydrolysis and DNA packaging. The eGFP-gp16 was successfully used as a marker for direct observation of DNA packaging and for direct counting of gp16 number in single molecule microscopy studies. Such engineering of gp16 will facilitate future studies on structure, function, distance, speed and motion mechanism of the phi29 DNA packaging motor by single molecule imaging.