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J. Biol. Chem., Vol. 278, Issue 34, 32300-32306, August 22, 2003

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Characterization of the Interactions within the mazEF Addiction Module of Escherichia coli^*

Junjie Zhang, Yonglong Zhang and Masayori Inouye 

From the Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

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)₆MazE 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)₆MazE 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 Leu⁵⁵ and Leu⁵⁸ 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.

Received for publication, May 7, 2003 , and in revised form, June 10, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Ln., Piscataway, NJ 08854. Tel.: 732-235-4115; E-mail: inouye{at}rwja.umdnj.edu.

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