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Journal of Bacteriology, September 1998, p. 4638-4643, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Functional Dissection of the Molybdate-Responsive Transcription Regulator, ModE, from Escherichia coli

Paul M. McNicholas, Mandy M. Mazzotta, Sabine A. Rech, and Robert P. Gunsalus*

Department of Microbiology and Molecular Genetics and the Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095-1489

Received 3 April 1998/Accepted 26 June 1998

The product of the Escherichia coli modE gene, ModE, is a member of a unique class of molybdate-responsive DNA binding proteins. Here we investigated the roles of the N- and C-terminal domains of ModE in mediating DNA binding and protein dimerization, respectively. Compared to the full-length protein, the N-terminal half of ModE has a greatly diminished capacity to bind the modA promoter in vitro and to repress expression from a modA-lacZ operon fusion in vivo. Fusing a protein dimerization domain, encoded by the C terminus of lambda  CI repressor protein, to the truncated ModE protein generated a ModE-CI fusion protein that not only displayed a greatly increased in vivo repressor activity but could also substitute for ModE at the moaA and dmsA promoters. In the reciprocal experiment, we restored repressor activity to a truncated CI protein by addition of the C-terminal domain of ModE, which is comprised of two MopI-like subdomains. By an in vivo competition assay, we also demonstrated that the CI-ModE chimeric protein retained the ability to interact with wild-type ModE. Finally, specific deletions within the ModE portion of the CI-ModE protein chimera abolished both in vivo repression and the ability to interact with wild-type ModE. Together, these data demonstrate that the N-terminal domain of ModE is sufficient to mediate DNA binding, although efficient binding requires that ModE form a dimer, a function that is supplied by the C-terminal MopI-like subdomains.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, 1602 Molecular Sciences Building, 405 Hilgard Avenue, University of California, Los Angeles, CA 90095. Phone: (310) 206-8201. Fax: (310) 206-5231. E-mail: robg{at}microbio.ucla.edu.

Journal of Bacteriology, September 1998, p. 4638-4643, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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