Characterization of the interactions within the mazEF addiction module of Escherichia coli
Characterization of the interactions within the mazEF addiction module of Escherichia coli
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DOI：
10.1074/jbc.m304767200
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发表时间：
2003-08-22
影响因子：
4.8
通讯作者：
Inouye, M
中科院分区：
文献类型：
--
作者：
Zhang, JJ;Zhang, YL;Inouye, M
在细菌中，程序性细胞死亡是通过一种名为“成瘾模块”的独特遗传系统来调节的，该系统由一对编码稳定毒素和不稳定抗毒素的基因组成。MazEF系统被认为是位于大肠杆菌染色体上的成瘾模块。MazF是一种稳定的毒素，而迷宫是一种不稳定的抗毒素，与MazF相互作用形成复合体。MAZE和MAZE-MazF复合体可以与mazEF启动子区域结合，调节mazEF的表达。在这里，我们证明了纯化的(His)(6)迷宫与mazEF启动子DNA的结合被MazF增强。迷宫N末端保守氨基酸残基(K7A、R8A、S12A和R16A)的定点突变破坏了(His)(6)迷宫和迷宫-MazF(His)(6)复合体的DNA结合能力，表明迷宫通过N-末端结构域与mazEF启动子DNA结合。在迷宫-MazF(His)(6)复合体中，迷宫和MazF(His)(6)的比例约为1：2。由于迷宫和MazF(His)(6)都以二聚体的形式存在，因此预测迷宫-MazF(His)(6)复合体(76.9 kDa)由一个迷宫二聚体和两个MazF(His)(6)二聚体组成。用酵母双杂交系统研究了迷宫与MazF的相互作用。结果表明，MazF与MazF的结合需要迷宫残基38~75区。该区域的定点突变显示Leu(55)和Leu(58)在迷宫-MazF复合体的形成中起重要作用，但在迷宫与mazEF启动子DNA的结合中不起作用。结果表明，迷宫由两个结构域组成，N端DNA结合域和C端与MazF相互作用。
In bacteria, programmed cell death is mediated through the unique genetic system called "addiction module," which consists of a pair of genes encoding a stable toxin and an unstable antitoxin. The mazEF system is known as an addiction module located on the Escherichia coli chromosome. MazF is a stable toxin, and MazE is a labile antitoxin interacting with MazF to form a complex. MazE and the MazE-MazF complex can bind to the mazEF promoter region to regulate the mazEF expression. Here we show that the binding of purified (His)(6)MazE to the mazEF promoter DNA was enhanced by MazF. The site-directed mutations at the conserved amino acid residues in MazE N-terminal region (K7A, R8A, S12A, and R16A) disrupted the DNA binding ability of both (His)(6)MazE and the MazE-MazF(His)(6) complex, suggesting that MazE binds to the mazEF promoter DNA through the N-terminal domain. The ratio of MazE to MazF(His)(6) in the MazE-MazF(His)(6) complex is about 1:2. Because both MazE and MazF(His)(6) exist as dimers by themselves, the MazE-MazF(His)(6) complex (76.9 kDa) is predicted to consist of one MazE dimer and two MazF(His)(6) dimers. The interaction between MazE and MazF was also characterized with the yeast two-hybrid system. It was found that the region from residues 38 to 75 of MazE was required for its binding to MazF. Site-directed mutagenesis at this region revealed that Leu(55) and Leu(58) play an important role in the MazE-MazF complex formation but not in MazE binding to the mazEF promoter DNA. The present results demonstrate that MazE is composed of two domains, the N-terminal DNA-binding domain and the C-terminal domain interacting with MazF.