Abstract
Tailed bacteriophages and herpes viruses use powerful molecular motors to translocate DNA into a preassembled prohead and compact the DNA to near crystalline density. The phage T4 motor, a pentamer of 70-kDa large terminase, gp17, is the fastest and most powerful motor reported to date. gp17 has an ATPase activity that powers DNA translocation and a nuclease activity that cuts concatemeric DNA and generates the termini of viral genome. An 18-kDa small terminase, gp16, is also essential, but its role in DNA packaging is poorly understood. gp16 forms oligomers, most likely octamers, exhibits no enzymatic activities, but stimulates the gp17-ATPase activity, and inhibits the nuclease activity. Extensive mutational and biochemical analyses show that gp16 contains three domains, a central oligomerization domain, and N- and C-terminal domains that are essential for ATPase stimulation. Stimulation occurs not by nucleotide exchange or enhanced ATP binding but by triggering hydrolysis of gp17-bound ATP, a mechanism reminiscent of GTPase-activating proteins. gp16 does not have an arginine finger but its interaction with gp17 seems to position a gp17 arginine finger into the catalytic pocket. gp16 inhibits DNA translocation when gp17 is associated with the prohead. gp16 restricts gp17-nuclease such that the putative packaging initiation cut is made but random cutting is inhibited. These results suggest that the phage T4 packaging machine consists of a motor (gp17) and a regulator (gp16). The gp16 regulator is essential to coordinate the gp17 motor ATPase, translocase, and nuclease activities, otherwise it could be suicidal to the virus.
Highlights
The structure of the phage T4 DNA packaging motor has been determined recently [7, 8]
Mutational and biochemical analyses established the catalytic residues in ATPase, translocase, and nuclease functional motifs that are critical for various steps of DNA packaging (14 –18)
Our results further show that gp16 stimulates the gp17ATPase activity by triggering the hydrolysis of gp17-bound ATP in a manner similar to that of the GTPase-activating proteins (GAPs). gp16 modulates gp17-dependent DNA translocation through interaction with the prohead-assembled gp17 complex. gp16 restricts gp17-nuclease such that the putative packaging initiation cut is made but extensive random DNA cutting is inhibited
Summary
Construction of g16 Mutant Clones—The g16 DNA fragments were amplified by PCR using purified phage T4 DNA as template and appropriate primers. The purified gp176 (0.2– 0.5 M) either alone or with gp (2–5 M) were incubated in a reaction mixture (20 l) containing 1 mM unlabeled ATP and 75 nM [␥32P]ATP (specific activity, 3000 Ci/mmol; GE Healthcare) at 37 °C in ATPase buffer (50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 5 mM MgCl2) for 20 min. Nuclease—The purified gp (0.2–1 M) was incubated either alone or in the presence of gp (0.2–10 M) with 100 ng of pAD10 plasmid DNA (29 kb) in a reaction mixture (25 l) containing 5 mM Tris-HCl, pH 8.0, 6 mM NaCl, and 5 mM MgCl2 for 15–30 min at 37 °C. The reaction was terminated by adding EDTA to a final concentration of 50 mM, and the samples were electrophoresed on 1.0% (w/v) agarose gel followed by ethidium bromide staining [13]
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