Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli.

Claverie-Martı́n, Félix; Magasanik, Boris

doi:10.1073/pnas.88.5.1631

Cited by 120 publications

(87 citation statements)

References 36 publications

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“…Perhaps IHF, upon binding (and bending) DNA, favourably presents the UAS(s) (psp enhancer) to the DNA-binding HTH motif of the activator. IHF does not influence the binding of the activator and E 54 holoenzyme in the glnHp2 and nifH promoters (Claverie-Martin and Magasanik, 1991;Santero et al, 1992).…”

Section: Discussionmentioning

confidence: 91%

“…IHF binding induces a sharp DNA bend (> 160Њ) (Rice et al, 1996) and can thus facilitate an interaction between proteins bound at distantly located elements. IHF does not stimulate transcription by itself (Goosen and Putte, 1995) and, in other promoters, does not affect the binding of either activator or E 54 holoenzyme to their respective DNA binding sites (Claverie-Martin and Magasanik, 1991;Santero et al, 1992). These earlier results are specific to the promoters studied; there is no reason in principle why the specific organization of regulatory elements in a 54 -dependent promoter region should not facilitate co-operative binding of the activator, IHF, and polymerase.…”

Section: Introductionmentioning

confidence: 78%

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PspF and IHF bind co‐operatively in the psp promoter‐regulatory region of Escherichia coli

Jovanović

Model

1997

Molecular Microbiology

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SummaryPspF bound to the psp enhancer activates E 54 holoenzyme-dependent transcription of the Escherichia coli phage-shock protein (psp ) operon and autogenously represses its own 70 -dependent transcription, thereby keeping its concentration at a low level. It has been demonstrated previously that integration host factor (IHF) bound to a DNA site located between the psp core promoter and the PspF binding sites stimulates psp expression. We show here that wild-type IHF strongly retards DNA containing the psp promoter region. In vitro, PspF binding to the psp enhancer facilitates IHF binding, while IHF binding to the pspFpspA-E promoter-regulatory region increases the efficacy of PspF binding to the upstream activating sequences (UASs). This is the first demonstration of co-operative binding of an activator and IHF in a 54 -dependent system. In the absence of IHF, in vivo autoregulation of pspF transcription is lifted and, consequently, PspF production is increased, indicating that IHF enhances PspF binding to the psp enhancer in vivo.

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Section: Discussionmentioning

confidence: 91%

Section: Introductionmentioning

confidence: 78%

PspF and IHF bind co‐operatively in the psp promoter‐regulatory region of Escherichia coli

Jovanović

Model

1997

Molecular Microbiology

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“…Both amino acid sequences could be aligned to that of the P subunit of E. coli IHF. On the basis of these two last amino acid sequences and by using the R. capsulatus codon usage, the following two oligonucleotides were synthesized: IA922, 5 (18), with a 5% (wt/vol) stacking gel and a 15% (wt/vol) resolving gel. For N-terminal sequence analysis, proteins were transferred to Problott membranes (Applied Biosystems) in a TE22 Mini Transphor unit (Hoefer) by the protocol recommended by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

Purification of the integration host factor homolog of Rhodobacter capsulatus: cloning and sequencing of the hip gene, which encodes the beta subunit

et al. 1993

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We describe a method for rapid purification of the integration host factor (IHF) homolog of Rhodobacter capsulatus that has allowed us to obtain microgram quantities of highly purified protein. R. capsulatus IHF is an a,3 heterodimer similar to IHF of Escherichia coli. We have cloned and sequenced the hip gene, which encodes the 0i subunit. The deduced amino acid sequence (10.7 kDa) has 46% identity with the I8 subunit of IHF from E. coli. In gel electrophoretic mobility shift DNA binding assays, R. capsulatus IHF was able to form a stable complex in a site-specific manner with a DNA fragment isolated from the promoter of the structural hupSL operon, which contains the IHF-binding site. The mutated IHF protein isolated from the Hup-mutant IR4, which is mutated in the himA gene (coding for the a subunit), gave a shifted band of greater mobility, and DNase I footprinting analysis has shown that the mutated IHF interacts with the DNA fragment from the hupSL promoter region differently from the way that the wild-type IHF does.The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus possesses a membrane-bound hydrogenase that functions in H2 uptake under physiological conditions (31). This enzyme enables R. capsulatus cells to grow autotrophically with H2 as the source of electrons (17). It is also synthesized under heterotrophic growth conditions, in particular in H2-evolving cultures in which hydrogenase synthesis is stimulated by H2 (9).The level of hydrogenase expression is closely coupled to environmental and growth conditions (9). Regulatory genes necessary for hydrogenase synthesis in R. capsulatus have recently been identified in the cluster of hup and hyp genes (8 (30,33) were grown anaerobically in the light in minimal salts (RCV) medium (15, 32) supplemented with DL-malate (30 mM) and L-glutamate (7 mM) as C and N sources, respectively, as previously described (7). DNA manipulations. Standard recombinant DNA techniques were performed as described previously (25). Plasmids pI,B2 and pI,B3 were constructed by inserting into the polylinker of pUC18 (34) the 5-kbp PstI-PstI and 2.2-kbp HindIll-HindIII fragments, respectively, of genomic R. capsulatus DNA that hybridized with the IHF13 probe. DNA sequencing on both strands was performed by the dideoxy chain termination method (26). DNA sequence analysis and homology searching were performed with LASERGENE programs (DNASTAR, Madison, Wis.). Gel retardation assays were performed as previously described (30), using the 274-bp EcoRI-EcoRI DNA fragment containing the IHF-binding site isolated from the hupS promoter.Purification of IHF. R capsulatus strains were grown anaerobically in 40-liter cultures until the A660 reached 1.2, typically yielding 120 g of wet cells. The cells were harvested by centrifugation and resuspended in 100 mM Tris HCl-20 mM EDTA, and 0.2 mg of lysosyme per ml was added. The cell extract (150 ml), obtained after sonication and ultracentrifugation (150,000 x g, 2 h) and containing 3 g of protein, was applied to a 5-ml Econo-Pac he...

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“…IHF plays a role in stimulation of transcription from the nifH and glnHp2 promoters by its ability to induce a bend in the DNA that brings the cr 4 RNA polymerase bound at the promoter in contact with an activator protein bound at a distant upstream site (6,12).…”

mentioning

confidence: 99%

A sigma 54 promoter and downstream sequence elements ftr2 and ftr3 are required for regulated expression of divergent transcription units flaN and flbG in Caulobacter crescentus

Mullin

Newton

1993

J Bacteriol

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In this study, we investigated the cis-acting sequences required for transcription of the divergent, cell cycle-regulated flaN and flbG operons of Caulobacter crescentus. Previous work showed that transcription of flbG in vivo depends on a cr& promoter and a sequence element calledftrl that is located about 100 bp upstream from the transcription start site (D. A. Mullin and A. Newton, J. Bacteriol. 171:3218-3227, 1989). We now show that regulation offlaN transcription in vivo depends on a o54 promoter and two ftr elements located downstream of the transcription start site at +86 (ftr2) and +120 (ftr3). Mutations in or between the conserved elements at -24 and -12 in this or4 promoter reduced or abolishedflaN transcription, and one mutation that eliminated flaN expression led to an increased level offlbG transcript. Mutations in ftr2 resulted in greatly reduced levels offlaN transcript but had no noticeable effect on flbG transcript levels. All three mutations constructed inftr3 resulted in elevated flaN and flbG transcript levels. We conclude thatftr2 is required for positive regulation offlaN, whereasftr3 appears to play a negative regulatory role inflaN andflbG expression.To explain the coordinated positive activation and negative autoregulation of these two transcription units and the effect of mutations on gene expression, we propose a model in which theflaN andflbG promoters interact through alternative DNA looping to form structures that are transcriptionally active or inactive.Flagellum biosynthesis in Caulobacter crescentus requires more than 50 flagellar (fla) genes, and about half of these are organized in transcription units that are located in three major clusters on the chromosome. These fla genes are under strict temporal regulation, as indicated by the observation that they are transcribed periodically in the cell cycle at about the time that the gene products assemble to form the flagellum. The precisely ordered sequence of fla gene expression during flagellum biosynthesis and the availability of powerful biochemical and genetic tools have made C. crescentus an attractive model for studying the mechanism of temporal gene expression during cell differentiation. A central focus of these studies has been to explain, on the molecular level, how genes are turned on and off in a defined sequence during the cell division cycle (for a review, see reference 26).The role of trans-acting genes in the expression of C. crescentus fla genes was indicated first by epistasis experiments (3, 31) and later examined in detail by experiments that measured the levels of specific fla gene transcripts in different nonmotile mutants (27). These studies suggested that the C. crescentus fla genes, like those in Eschenchia coli (14, 15) and Salmonella typhimurium (18), are organized into a regulatory hierarchy in which the expression of fla genes at each level depends on the expression of genes above them in the hierarchy (27,38 identified, but there is evidence that a step in the DNA synthesis pathway is required for the...

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Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli.

Cited by 120 publications

References 36 publications

PspF and IHF bind co‐operatively in the psp promoter‐regulatory region of Escherichia coli

PspF and IHF bind co‐operatively in the psp promoter‐regulatory region of Escherichia coli

Purification of the integration host factor homolog of Rhodobacter capsulatus: cloning and sequencing of the hip gene, which encodes the beta subunit

A sigma 54 promoter and downstream sequence elements ftr2 and ftr3 are required for regulated expression of divergent transcription units flaN and flbG in Caulobacter crescentus

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