Functional dissection of the molybdate-responsive transcription regulator, ModE, from Escherichia coli.
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.
Department of Microbiology and Molecular Genetics and the Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095-1489, USA.
P M
PM
McNicholas
M M
MM
Mazzotta
S A
SA
Rech
R P
RP
Gunsalus
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D017931:DNA Primers; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C491064:ModE protein, E coli; C101050:ModE protein, bacteria; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C044659:molybdate; D008982:Molybdenum