Identification of the Bases in theompFRegulatory Region, which Interact with the Transcription Factor OmpR

Huang, Ke-Jung; Igo, Michèle M.

doi:10.1006/jmbi.1996.0540

Cited by 95 publications

(121 citation statements)

References 32 publications

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“…These DNA fragments (Fig. 1B) take into account the contact information obtained from our DNA footprinting experiments (6). That the DNA fragments used in this study have different endpoints when compared with the DNA fragments used in earlier studies seems the simplest explanation for any disagreement between our results and those obtained by others (7,8,12,13).…”

Section: Resultsmentioning

confidence: 53%

“…These studies indicated that each individual OmpR binding site spans Ϸ18 bp and contains two half-sites oriented as asymmetric direct repeats. Analysis of the individual sites at ompF also established that only the F1 site is capable of forming a stable OmpR͞DNA complex as an isolated site (6)(7)(8)12) and that two OmpR molecules are required for stable binding to this site (6). Therefore, although OmpR exists in solution as a monomer under most conditions that have been examined (5,14,15), OmpR is unable to form stable protein͞DNA complexes as a monomer.…”

Section: Resultsmentioning

confidence: 98%

“…For this experiment, we compared the effects of phosphorylation on the binding of OmpR to a DNA fragment containing the wild-type F1, F2, and F3 sites vs. a DNA fragment containing a mutant F2 site (F2*) between flanking wild-type F1 and F3 sites. Our strategy for inactivating the F2 site was derived from our DNA footprinting analysis of the ompF regulatory region (6). These studies identified a G residue in the left half-site that was highly conserved among all OmpR-binding sites.…”

Section: Resultsmentioning

confidence: 99%

“…4, the DNA fragments F1-F2-F3 (IKH23) and F1-F2*-F3 (IKH24) were generated by PCR from the plasmid pKJH6 (6). The DNA fragment F1-F2-F3 contains the wild-type sequence from Ϫ99 to Ϫ35.…”

Section: Dna Fragments Used In the Dna Migration Retardationmentioning

confidence: 99%

“…The DNA migration retardation assays were performed as described (6) using the proteins and DNA fragments indicated in the figure legends. Wild-type OmpR and maltose binding protein (MBP)-EnvZ fusion protein were overexpressed and purified as described (6). The OmpR D55Q mutant protein was overexpressed using plasmid pLAN202 (unpublished results) and then purified using the protocol outlined previously for wild-type OmpR (6).…”

Section: Dna Fragments Used In the Dna Migration Retardationmentioning

confidence: 99%

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Phosphorylation stimulates the cooperative DNA-binding properties of the transcription factor OmpR

Huang

Lan

Igo

1997

Proc. Natl. Acad. Sci. U.S.A.

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The two-component regulatory proteins OmpR and EnvZ of Escherichia coli K-12 regulate expression of the major outer membrane porin protein, OmpF. OmpR is a DNA-binding protein that is involved in both the positive and negative control of ompF transcription. EnvZ is a histidine kinase that phosphorylates OmpR in response to environmental signals. We used DNA migration retardation analysis to examine the interactions of OmpR and the phosphorylated form of OmpR (OmpR-P) with the regulatory region immediately upstream of the ompF promoter. Our results indicate that the binding of OmpR to this regulatory region is cooperative and that phosphorylation significantly stimulates these cooperative interactions. Moreover, although phosphorylation increases the intrinsic binding of OmpR to a single OmpR-binding site, the primary role of phosphorylation in ompF regulation is to facilitate cooperative interactions between OmpR molecules bound at adjacent sites. Based on these results, we propose a model to explain how the phosphorylation of OmpR could stimulate the occupancy of specific sites in the ompF regulatory region, thereby resulting in the activation or repression of ompF transcription under the appropriate environmental conditions. Expression of the major outer membrane porin proteins OmpF and OmpC of Escherichia coli K-12 is regulated in response to a wide variety of environmental signals (reviewed in refs. 1-3). The most extensively studied of these signals is the osmolarity of the growth medium. At low osmolarity, the OmpF porin is preferentially expressed and only low levels of the OmpC porin are found. Conversely, at high osmolarity, OmpC is preferentially expressed and only low levels of OmpF are found. This fluctuation in porin expression is controlled at the transcriptional level by the two-component regulatory system, EnvZ and OmpR. EnvZ is a membrane-bound histidine kinase that modulates the activity of the DNA-binding protein OmpR via phosphorylation (refs. 2 and 3 and references therein). The phosphorylated form of OmpR (OmpR-P), which is the active form of the protein, is a transcriptional activator at the ompC promoter and can function as either an activator or a repressor at the ompF promoter (4).According to the current model (3), at low osmolarity the cellular concentration of OmpR-P is low. This level is sufficient for OmpR-P to interact with the sites in the ompF regulatory region that are responsible for activating ompF transcription. At high osmolarity, the cellular concentration of OmpR-P is much greater. As a result of this increase, OmpR-P is now capable of occupying the sites in the ompC regulatory region responsible for activating ompC transcription, and the sites at ompF responsible for repressing ompF transcription. Thus, this model predicts that the regulation of ompF and ompC is a direct consequence of the level of OmpR-P in the cell and is dependent on the way in which OmpR-P interacts with sites in the ompF and ompC regulatory regions.A number of studies have addressed the basi...

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Section: Resultsmentioning

confidence: 53%

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

“…4, the DNA fragments F1-F2-F3 (IKH23) and F1-F2*-F3 (IKH24) were generated by PCR from the plasmid pKJH6 (6). The DNA fragment F1-F2-F3 contains the wild-type sequence from Ϫ99 to Ϫ35.…”

Section: Dna Fragments Used In the Dna Migration Retardationmentioning

confidence: 99%

Section: Dna Fragments Used In the Dna Migration Retardationmentioning

confidence: 99%

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