Characterization of ATPase activity of a defined in vitro system for packaging of bacteriophage T3 DNA.

We have developed a defined in vitro system for packaging phage T3 DNA which is composed of purified proheads and the noncapsid proteins gp18 and gp19, products of genes 18 and 19 (K. Hamada, H. Fujisawa, and T. Minagawa, 1986, Virology 151, 119-123). The in vitro system displayed an ATPase activity. The requirements for ATPase activity were the same as those for DNA packaging. ATPase was inhibited by a nonhydrolyzable ATP analog, adenosine-5'-O-(3'-thiotriphosphate) (ATP-gamma-S). ATPase activity did not display specificity for T3 DNA. A reaction mixture containing 8- proheads, proheads deficient in gp8, a portal protein for DNA entrance, or mature heads had no gp18- gp19-dependent ATPase activity. gp8 itself had no ATPase activity and did not complement 8- proheads for ATPase activity. Photoaffinity labeling of proheads, gp18 and gp19 with 8-azidoadenosine-5'-[alpha-32P]triphosphate([32P]8-N3ATP) resulted in preferential labeling of gp19. Protection from incorporation of [32P]8-N3ATP was afforded by ATP but not by AMP and ADP. From these results, it is concluded that gp19 has an ATP binding site(s). A conserved sequence of ATP-binding site containing Gly-X-Gly-X-X-Gly-X-Val is found in gp19.