The DNA translocating ATPase of bacteriophage T4 packaging motor.
In double-stranded DNA bacteriophages the viral DNA is translocated into an empty prohead shell by a powerful ATP-driven motor assembled at the unique portal vertex. Terminases consisting of two to three packaging-related ATPase sites are central to the packaging mechanism. But the nature of the key translocating ATPase, stoichiometry of packaging motor, and basic mechanism of DNA encapsidation are poorly understood. A defined phage T4 packaging system consisting of only two components, proheads and large terminase protein (gp17; 70 kDa), is constructed. Using the large expanded prohead, this system packages any linear double-stranded DNA, including the 171 kb T4 DNA. The small terminase protein, gp16 (18 kDa), is not only not required but also strongly inhibitory. An ATPase activity is stimulated when proheads, gp17, and DNA are actively engaged in the DNA packaging mode. No packaging ATPase was stimulated by the N-terminal gp17-ATPase mutants, K166G (Walker A), D255E (Walker B), E256Q (catalytic carboxylate), D255E-E256D and D255E-E256Q (Walker B and catalytic carboxylate), nor could these sponsor DNA encapsidation. Experiments with the two gp17 domains, N-terminal ATPase domain and C-terminal nuclease domain, suggest that terminase association with the prohead portal and communication between the domains are essential for ATPase stimulation. These data for the first time established an energetic linkage between packaging stimulation of N-terminal ATPase and DNA translocation. A core pathway for the assembly of functional DNA translocating motor is proposed. Since the catalytic motifs of the N-terminal ATPase are highly conserved among >200 large terminase sequences analyzed, these may represent common themes in phage and herpes viral DNA translocation.
Kondabagil
Kiran R
KR
Department of Biology, The Catholic University of America, Washington, DC 20064, USA.
Zhang
Zhihong
Z
Rao
Venigalla B
VB
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology*; D042003:DNA Packaging / Q000235:genetics*; D004279:DNA, Viral / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism
D004279:DNA, Viral; D050505:Mutant Proteins; D014764:Viral Proteins; C065038:gp17 protein, Enterobacteria phage T4; D004706:Endodeoxyribonucleases; C030894:terminase; D000251:Adenosine Triphosphatases