PMID:9721306
| Citation |
McNicholas, PM, Mazzotta, MM, Rech, SA and Gunsalus, RP (1998) Functional dissection of the molybdate-responsive transcription regulator, ModE, from Escherichia coli. J. Bacteriol. 180:4638-43 |
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| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Bacterial Proteins; Base Sequence; DNA Primers; Dimerization; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Molecular Sequence Data; Molybdenum/metabolism; Promoter Regions, Genetic; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/metabolism; Sequence Homology, Amino Acid; Transcription Factors/chemistry; Transcription Factors/metabolism |
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