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Functional analysis of the DNA-packaging/terminase protein gp17 from bacteriophage T4
1Department of Biology, The Catholic University of America, Washington, DC, 20064, USA.
Journal of Molecular Biology
|August 28, 1998
Summary
Bacteriophage T4 gp17's metal-binding motif, specifically involving histidine residues H411-H414 and H430-H436, is crucial for DNA packaging and terminase functions. Mutations in this motif severely impair these essential processes for phage replication.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bacteriophage T4 uses a terminase complex, primarily the large subunit gp17, for ATP-driven DNA packaging into its capsid.
- The gp17 subunit possesses multiple functional sites, including DNA cleavage, portal binding, DNA packaging, and ATPase activity, suggesting a complex mechanism of action.
Purpose of the Study:
- To investigate the functional significance of potential metal-binding motifs within the bacteriophage T4 gp17 subunit.
- To elucidate the mechanism underlying the essential DNA packaging and terminase activities of gp17.
Main Methods:
- An expression-based selection strategy was employed to identify mutants with defective terminase function.
- Site-directed mutagenesis was used to introduce mutations in conserved histidine residues within putative metal-binding motifs of gp17.
- Functional assays were performed to assess the impact of mutations on terminase and DNA-packaging activities.
Main Results:
- A single H436R mutation in gp17 resulted in a significant loss of both terminase and DNA-packaging functions, proving lethal to T4 development in vivo.
- Mutations within the histidine-rich region, specifically at the H411 and H430 pairs, abolished gp17 function, supporting their role in a metal-binding motif.
- Mutations in the H382 pair showed no effect, while substitutions at K166 in an ATP-binding site also impaired function.
Conclusions:
- A metal-binding motif involving histidine residues (H411-H414 and H430-H436) is essential for the function of bacteriophage T4 gp17.
- Metal-terminase interactions are likely critical for the structural alignment and stabilization of functional sites in T4 terminase and other double-stranded DNA phage terminases.