Purification and functional characterization of p16, the ATPase of the bacteriophage Phi 29 packaging machinery

Bacteriophage Phi 29 codes for a protein (p16) that is required for viral D NA packaging both in vivo and in vitro. Co-expression of p16 with the chape ronins GroEL and GroES has allowed its purification in a soluble form. Puri fied p16 shows a weak ATPase activity that is stimulated by either DNA or R NA, irrespective of the presence of any other viral component. The stimulat ion of ATPase activity of p16, although induced under packaging conditions, is not dependent of the actual DNA packaging and in this respect the Phi 2 9 enzyme is similar to other viral terminases. Protein p16 competes with DN A and RNA in the interaction with the viral prohead, which occurs through t he N-terminal region of the connector protein (p10). In fact, p16 interacts in a nucleotide-dependent fashion with the viral Phi 29-encoded RNA (pRNA) involved in DNA packaging, and this binding can be competed with DNA. Our results are consistent with a model for DNA translocation in which p16, bou nd and organized around the connector, acts as a power stroke to pump the D NA into the prohead, using the hydrolysis of ATP as an energy source.