A distant upstream site involved in the negative regulation of the Escherichia coli ompF gene

Huang, Ke-Jung; Schieberl, J. L.; Igo, Michèle M.

doi:10.1128/jb.176.5.1309-1315.1994

Cited by 56 publications

(64 citation statements)

References 52 publications

(27 reference statements)

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“…Finally, it seems likely that the stimulatory effect of phosphorylation on cooperative interactions could also be used to explain ompF repression under high osmolarity conditions. Previous studies have suggested that the repression of ompF transcription involves the formation of a DNA loop (8,18,19). One intriguing possibility is that the stimulation of cooperative interaction by high cellular levels of OmpR-P could result in the occupancy of the F3 and F4 sites.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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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“…38 It has been reported that the difference in OmpF expression between the E. coli BL21(DE3) and K12 strain might be explained by two mutations at positions -18 and -363 in the ompF promoter and its upstream region. Position -363 is within the OmpR binding site, 39 so the mutation seems to affect OmpR binding, and thus the repression of ompF expression. We have prepared a BL21(DE3) ompF knock-out strain for protein expression for a future comparison study.…”

Section: Crystal Packing Of Ompf Prepared In Fc12mentioning

confidence: 99%

Structures of the OmpF porin crystallized in the presence of foscholine‐12

et al. 2010

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The endogenous Escherichia coli porin OmpF was crystallized as an accidental byproduct of our efforts to express, purify, and crystallize the E. coli integral membrane protein KdpD in the presence of foscholine-12 (FC12). FC12 is widely used in membrane protein studies, but no crystal structure of a protein that was both purified and crystallized with this detergent has been reported in the Protein Data Bank. Crystallization screening for KdpD yielded two different crystals of contaminating protein OmpF. Here, we report two OmpF structures, the first membrane protein crystal structures for which extraction, purification, and crystallization were done exclusively with FC12. The first structure was refined in space group P21 with cell parameters a 5 136.7 Å , b 5 210.5 Å , c 5 137 Å , and b 5 100.5°, and the resolution of 3.8 Å . The second structure was solved at the resolution of 4.4 Å and was refined in the P321 space group, with unit cell parameters a 5 215.5 Å , b 5 215.5 Å , c 5 137.5 Å , and c 5 120°. Both crystal forms show novel crystal packing, in which the building block is a tetrahedral arrangement of four trimers. Additionally, we discuss the use of FC12 for membrane protein crystallization and structure determination, as well as the problem of the OmpF contamination for membrane proteins overexpressed in E. coli.

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“…Purified GST-Tas was incubated with 32P-end labeled oligonucleotides or DNA fragments in a binding buffer (10 mM Tris-Cl (pH 7.5)/65 mM KCl/2.5 mM MgCI2/l mM dithiothreitol/5% glycerol) con- Footprinting by the DNase I Protection Method. The DNase I protection assay for identifying nucleotides in viral target elements binding Tas was done as described (18,20). Briefly, probes were prepared by digesting the plasmid pMl (see Fig.…”

Section: Methodsmentioning

confidence: 99%

The transcriptional transactivator of simian foamy virus 1 binds to a DNA target element in the viral internal promoter.

Zou¹,

Luciw²

1996

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

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The transcriptional transactivator (Tas) of simian foamy virus type 1 strongly augments gene expression directed by both the promoter in the viral long terminal repeat and the newly discovered internal promoter located within the env gene. A region of 121 bp, located immediately 5' to the TATA box in the internal promoter, is required for transactivation by Tas. The present study aimed to identify the precise Tas-responsive target(s) in this region and to determine the role of Tas in transcriptional regulation. By analysis of both clustered-site mutations and hybrid promoters in transient expression assays in murine and simian cells, two separate sequence elements within this 121-bp region were shown to be Tas-dependent transcriptional enhancers. These targets, each <30 bp in length and displaying no apparant sequence homology one to the other, are designated the promoterproximal and promoter-distal elements. By means of the gel electrophoresis mobility-shift assay, using purified glutathione S-transferase-Tas fusion protein expressed in Escherichia coli, the target proximal to the TATA box exhibited strong binding to glutathione S-transferase-Tas, whereas the distal element appears not to bind. In addition, footprint analysis revealed that 26 bp in the promoter proximal element was protected by glutathione S-transferase-Tas from DNase I. We propose a model for transactivation of the simian foamy virus type 1 internal promoter in which Tas interacts directly with the proximal target element positioned immediately 5' to the TATA box. In this model, Tas attached to this element is presumed to interact with a component(s) of the cellular RNA polymerase II initiation complex and thereby enhance transcription directed by the viral internal promoter.Foamy viruses, members of the spumavirus genus of retroviruses, have complex genomes encoding virion structural genes, a transcriptional transactivator, and one or two additional open reading frames (ORFs) of undefined function (1-3). Human foamy virus (HFV) and the simian foamy viruses (SFVs) contain two promoters for initiation of viral transcription. One promoter is located in the 5' long terminal repeat (LTR) and produces the full-length viral transcript, which is the precursor to spliced subgenomic transcripts (4). Another promoter is embedded in sequences within the 3' portion of the envelope gene and thus directs transcription of the viral transactivator and the ORF(s) (5-7). The transactivator has been designated taf for SFV (8, 9) and bel-i for HFV (10, 11).At the First International Foamy Virus Meeting (November 1994, London), it was recommended that taf and bel-i be designated tas (transactivator of spumavirus).Transient expression assays in several cell types have revealed that SFV and HFV transcriptional transactivator (Tas) transactivates both the homologous viral LTR and the internal promoter. Transactivation is mediated through target elements in the U3 domain of the LTR (9, 12-15) and sequences immediately 5' to the TATA box in the internal promote...

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A distant upstream site involved in the negative regulation of the Escherichia coli ompF gene

Cited by 56 publications

References 52 publications

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

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

Structures of the OmpF porin crystallized in the presence of foscholine‐12

The transcriptional transactivator of simian foamy virus 1 binds to a DNA target element in the viral internal promoter.

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