The N-terminal ATPase site in the large terminase protein gp17 is critically required for DNA packaging in bacteriophage T4.
Double-stranded DNA packaging in bacteriophages is apparently driven by the most powerful molecular motor ever measured. Although it is widely accepted that a translocating ATPase powers the DNA packaging machine, the identity of the ATPase that generates this driving force is unknown. Evidence suggests that the large terminase protein gp17, which possesses two consensus ATP binding motifs and an ATPase activity, is a strong candidate for the translocating ATPase in bacteriophage T4. This hypothesis was tested by a PCR-directed combinatorial mutagenesis approach in which mutant libraries consisting of all possible codon combinations were constructed at the signature residues of the ATP binding motifs. The impact on gp17 function of each randomly selected mutant was evaluated by phenotypic analysis following recombinational transfer into the viral genome. The precise mutation giving rise to a particular phenotype was determined by DNA sequencing. The data showed that the N-terminal ATP binding site I (SRQLGKT(161-167)), but not the ATP binding site II (TAAVEGKS(299-306)), is critical for gp17 function. Even conservative substitutions such as G165A, K166R, and T167A were not tolerated at the GKT signature residues, which are predicted to interact with the ATP substrate. Biochemical analyses of the mutants showed a complete loss of in vitro DNA packaging activity but not the terminase (DNA-cutting) activity. The purified K166G mutant showed a loss of gp17-ATPase activity. The data, for the first time, implicated a specific ATPase center in the viral dsDNA packaging.
Rao
V B
VB
Department of Biology, The Catholic University of America, Washington, DC 20064, USA. rao@cua.edu
Mitchell
M S
MS
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D019065:Virus Assembly
D004279:DNA, Viral; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; C104812:terminase, phage T4; D000251:Adenosine Triphosphatases; D008239:Lysine