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PMID:3294420
| Citation |
Rao, VB and Black, LW (1988) Cloning, overexpression and purification of the terminase proteins gp16 and gp17 of bacteriophage T4. Construction of a defined in-vitro DNA packaging system using purified terminase proteins. J. Mol. Biol. 200:475-88 |
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| Abstract |
Terminases of double-stranded DNA bacteriophages are required for packaging and generation of terminii in replicated concatemeric DNA molecules. Genetic evidence suggests that these functions in phage T4 are carried out by the products of genes 16 and 17. We cloned these T4 genes into a heat-inducible cI repressor-lambda PL promoter vector system, and overexpressed them in Escherichia coli. We developed an in-vitro DNA packaging system, which, consistent with the genetic data, shows an absolute requirement for the terminase proteins. The overexpressed terminase proteins gp16 and gp17 appear to form a specific complex and an ATP binding site is present in the gp17 molecule. We purified the terminase proteins either as individual gp16 or gp17 proteins, or as a gp16-gp17 complex. The gp16 function of the terminase complex is dispensable for packaging mature DNA, whereas gp17 is essential for packaging DNA under any condition tested. We constructed a defined in-vitro DNA packaging system with the purified terminase proteins, purified proheads and a DNA-free phage completion gene products extract. All the components of this system can be stored at -90 degrees C without loss of packaging activity. The terminase proteins, therefore, may serve as useful reagents for mechanistic studies on DNA packaging, as well as to develop T4 as a packaging-cloning vector. |
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| Keywords |
Cloning, Molecular; DNA, Viral; Endodeoxyribonucleases/genetics; Endodeoxyribonucleases/isolation & purification; Escherichia coli/genetics; T-Phages/genetics; Viral Proteins/genetics |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
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Contributes to |
GO:0043493: viral terminase complex |
ECO:0000314: |
C |
gp16 and gp17 Terminase genes from T4 were over expressed in Ecoli bacteria in the presence of empty phage proheads in order to asses the headful DNA packaging abilities of the genes. Gp16 and gp17 were expressed individually and together in a complex. It was found that, "the gp16 function of the terminase complex is dispensable for packaging mature DNA, whereas gp17 is essential for packaging DNA under any condition tested." It was therefore determined that gp17 is the main Terminase protein in T4 necessary for the molecular function of DNA packaging. |
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Notes
See also
References
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