The linker region between the helicase and primase domains of the bacteriophage T7 gene 4 protein is critical for hexamer formation
The linker region between the helicase and primase domains of the bacteriophage T7 gene 4 protein is critical for hexamer formation
复制标题
DOI：
10.1074/jbc.274.42.30303
复制
发表时间：
1999-10-15
影响因子：
4.8
通讯作者：
Richardson, CC
中科院分区：
文献类型：
--
作者：
Guo, SY;Tabor, S;Richardson, CC
噬菌体T7的基因4蛋白是一种功能性六聚体，具有DNA解旋酶和引发酶活性。这两种活性都依赖于蛋白质在与dTTP水解偶联的反应中沿沿着DNA的单向运动。我们已经表征了全长基因4蛋白（gp 4）以及从残基219开始的三个羧基末端片段的dTTP活性和六聚体形成位于引发酶和解旋酶结构域之间的残基242和271之间的区域对于基因4蛋白的寡聚化是关键的。在天然凝胶电泳过程中，gp 4、gp 4-C219和gp 4-C241作为寡聚体迁移，而gp 4-C272是单体。gp 4-C272的dTTR活性的稳态k（cat）随蛋白质浓度急剧增加，表明其仅在高浓度下形成寡聚体。gp 4-C219和gp 4-C241均与gp 4形成稳定的复合物，而gp 4-C272仅与gp 4弱相互作用。表面等离子体共振的测量表明T7 DNA聚合酶的单体结合gp 4、gp 4-C219或gp 4-C241的二聚体，但结合gp 4-C272的单体。基因4蛋白的寡聚化结构域与NTP结合结构域的氨基末端相邻。
The gene 4 protein of bacteriophage T7, a functional hexamer, comprises DNA helicase and primase activities. Both activities depend on the unidirectional movement of the protein along single-stranded DNA in a reaction coupled to the hydrolysis of dTTP, We have characterized dTTPase activity and hexamer formation for the full-length gene 4 protein (gp4) as well as for three carboxyl-terminal fragments starting at residues 219 (gp4-C219), 241 (gp4-C241), and 272 (gp4-C272), The region between residues 242 and 271, residing between the primase and helicase domains, is critical for oligomerization of the gene 4 protein. A functional TPase active site is dependent on oligomerization, During native gel electrophoresis, gp4, gp4-C219, and gp4-C241 migrate as oligomers, whereas gp4-C272 is monomeric, The steady-state k(cat) for dTTPase activity of gp4-C272 increases sharply with protein concentration, indicating that it forms oligomers only at high concentrations. gp4-C219 and gp4-C241 both form a stable complex with gp4, whereas gp4-C272 interacts only weakly with gp4, Measurements of surface plasmon resonance indicate that a monomer of T7 DNA polymerase binds to a dimer of gp4, gp4-C219, or gp4-C241 but to a monomer of gp4-C272, Like the homologous RecA and F-1-ATPase proteins, the oligomerization domain of the gene 4 protein is adjacent to the amino terminus of the NTP-binding domain.