Biochemical characterization of an ATPase activity associated with the large packaging subunit gp17 from bacteriophage T4.
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 Ni(2+)-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.
Leffers
G
G
Department of Biology, The Catholic University of America, Washington, DC 20064, USA.
Rao
V B
VB
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D004279:DNA, Viral / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000096:biosynthesis / Q000378:metabolism*; D004926:Escherichia coli; D008970:Molecular Weight; D011487:Protein Conformation; D014764:Viral Proteins / Q000096:biosynthesis / Q000378:metabolism*
D004279:DNA, Viral; D014764:Viral Proteins; D004706:Endodeoxyribonucleases; C104812:terminase, phage T4; D000251:Adenosine Triphosphatases