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J. Biol. Chem., Vol. 275, Issue 47, 37127-37136, November 24, 2000
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From the Department of Biology, The Catholic University of America,
Washington, D. C. 20064
Double-stranded DNA-packaging in icosahedral
bacteriophages is believed to be driven by a packaging "machine"
constituted by the portal protein and the two packaging/terminase
proteins assembled at the unique portal vertex of the empty prohead
shell. Although ATP hydrolysis is evidently the principal driving
force, which component of the packaging machinery functions as the
translocating ATPase has not been elucidated. Evidence suggests that
the large packaging subunit is a strong candidate for the translocating ATPase. We have constructed new phage T4 terminase recombinants under
the control of phage T7 promoter and overexpressed the
packaging/terminase proteins gp16 and gp17 in various configurations.
The hexahistidine-tagged-packaging proteins were purified to near
homogeneity by Ni2+-agarose chromatography and were
shown to be highly active for packaging DNA in vitro. The
large packaging subunit gp17 but not the small subunit gp16 exhibited
an ATPase activity. Although gp16 lacked ATPase activity, it enhanced
the gp17-associated ATPase activity by >50-fold. The gp16 enhancement
was specific and was due to an increased catalytic rate for ATP
hydrolysis. A phosphorylated gp17 was demonstrated under conditions of
low catalytic rates but not under high catalytic rates in the presence
of gp16. The data are consistent with the hypothesis that a weak ATPase
is transformed into a translocating ATPase of high catalytic capacity after assembly of the packaging machine.
Biochemical Characterization of an ATPase Activity Associated
with the Large Packaging Subunit gp17 from Bacteriophage T4*
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 202-319-5271;
Fax: 202-319-6161; E-mail: rao@cua.edu.
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