Functional analysis of the DNA-packaging/terminase protein gp17 from bacteriophage T4.
In bacteriophage T4, the terminase complex constituted by the large subunit gp17 (69 kDa) and the small subunit gp16 (18 kDa) is a critical component of the ATP-driven DNA-packaging pump that translocates DNA into an empty capsid shell. Evidence suggests that the large subunit gp17 is the critical component and consists of a number of the functional sites required for DNA-packaging. It exhibits a terminase activity that introduces non-specific cuts into DNA, a portal vertex binding site that allows linkage of cleaved DNA to an empty prohead, an in vitro DNA-packaging activity, and an ATPase activity. In addition, a consensus metal-binding motif and two consensus ATP-binding sites have been identified by sequence analysis. In order to understand the mechanism of action of the multifunctional gp17, we developed an expression-based selection strategy to select for mutants that are defective in terminase function. Characterization of one of the mutants revealed a unique phenotype in which a single H436R mutation resulted in a dramatic loss of both the terminase and the DNA-packaging functions. Indeed, in vivo substitution of H436 with any of the 12 amino acids for which a suppressor is available was lethal to T4 development. According to one hypothesis, H436 is part of a metal-binding motif that is essential for gp17 function. This hypothesis was tested by introducing mutations at each of the three histidine pairs, the H382-X2-H385 pair, the H411-X2-H414 pair and the H430-X5-H436 pair, which constitute the histidine-rich region near the C terminus of gp17. A mutation at either the H411 pair or the H430 pair resulted in a loss of gp17 function, whereas a mutation at the H382 pair had no effect. In addition to the putative metal-binding motif, substitutions at residue K166 within the putative N terminus-proximal ATP-binding site also resulted in a loss of gp17 function. We propose that a metal-binding motif involving the histidine residues within the sequence H411-X2-H414-X15-H430-X5-H436 is essential for gp17 function. Metal-terminase interactions may be required for structural alignment and stabilization of functional sites in phage T4 terminase and other double-stranded DNA phage terminases.
Department of Biology, The Catholic University of America, Washington, DC, 20064, USA.
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D000255:Adenosine Triphosphate / Q000378:metabolism; D017122:Bacteriophage T4 / Q000201:enzymology*; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000235:genetics; D008670:Metals / Q000378:metabolism; D008854:Microscopy, Electron; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation; D014764:Viral Proteins / Q000737:chemistry
D004279:DNA, Viral; D008670:Metals; D014764:Viral Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; C104812:terminase, phage T4