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At least 109 records · Page 6Linked to original sources
PubMed1993

Expression of the gene encoding the major bacterial nucleotide protein H-NS is subject to transcriptional auto-repression.

Expression of a promoterless cat gene fused to a DNA fragment of approximately 400 bp, beginning at -313 of Escherichia coli hns, was significantly repressed in E. coli and Salmonella typhimurium strains with wild-type hns but not in mutants carrying hns alleles. CAT expression from fusions containing a shorter (110 bp) segment of hns was essentially unaffected in the same genetic backgrounds. The stage of growth was found to influence the extent of repression which was maximum (approximately 75%) in mid-log cultures and negligible in cells entering the stationary phase. The level of repression in early-log phase was lower than in mid-log phase cultures, probably because of the presence of high levels of Fis protein, which counteracts the H-NS inhibition by stimulating hns transcription. The effects observed in vivo were mirrored by similar results obtained in vitro upon addition of purified H-NS and Fis protein to transcriptional systems programmed with the same hns-cat fusions. Electrophoretic gel shift assays, DNase I footprinting and cyclic permutation gel analyses revealed that H-NS binds preferentially to the upstream region of its own gene recognizing two rather extended segments of DNA on both sides of a bend centred around -150. When these sites are filled by H-NS, an additional site between approximately -20 and -65, which partly overlaps the promoter, is also occupied. Binding of H-NS to this site is probably the ultimate cause of transcriptional auto-repression.

Bacterial Outer Membrane Proteins↗
PubMed1990

Sequences upstream of the-35 hexamer of rrnB P1 affect promoter strength and upstream activation.

Transcription from Escherichia coli ribosomal RNA promoters is increased about 20-fold in vivo by a DNA sequence (the Upstream Activation Region, UAR) located upstream of the -35 conserved hexamer. The UAR stimulates transcription through two mechanisms: one which involves binding of the Fis protein to the UAR, and another mechanisms which functions in the absence of additional protein factors. We have previously constructed a collection of mutations in the region upstream of the -35 hexamer of rrnB P1. Most of these mutations have either no effect on promoter activity or decrease activity 2-5-fold in vivo (Gaal, T., Barkei, J., Dickson, R.R., De Boer, H.A., De Haseth, P.L., Alavi, H. and Gourse, R.L.(1989) J. Bacteriol. 171, 4852-4861). Two mutations leave both the -35 consensus hexamer and the Fis binding consensus sequence intact, yet have larger (14-50-fold) effects on transcription. One substitution just upstream of the -35 hexamer (a C to T change at position -37) primarily affects intrinsic promoter strength, leaving the UAR functional. On the other hand, a three base pair deletion (bases -38 through -40) severely reduces UAR-mediated activity. A substitution covering the three base pair deletion was constructed and found to be activated normally. UAR function appears dependent on its position relative to the RNA polymerase binding site, suggesting that a particular spatial geometry may be necessary for Fis-dependent and/or factor-independent activation to occur.

Base Sequence↗
PubMed1990

E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo.

An upstream activation region (UAR) contributes to the extremely high activity of the Escherichia coli ribosomal RNA promoter, rrnB P1, increasing its activity 20- to 30-fold over that of the same promoter lacking the UAR. We have used DNase footprinting to define three specific sites in the rrnB P1 UAR that bind Fis, a protein identified previously by its role in recombinational enhancer function in other systems. We find that purified Fis activates transcription from promoters containing these sites 10- to 20-fold in vitro at concentrations correlating with the filling of these sites. Three approaches indicate that Fis contributes to the function of the UAR in vivo. First, there is a progressive loss in the activity of rrnB P1-lacZ fusions as Fis binding sites are deleted. Second, an rrnB P1 promoter with a mutation in a Fis binding site has 5-fold reduced transcription activity in vivo, dramatically reduced Fis binding in vitro, and shows no Fis dependent transcription activation in vitro. Third, upstream activation is reduced 5-fold in a Fis- strain. We show that rRNA promoters derepress in response to the loss of Fis in vivo in accord with the predictions of the negative feedback model for rRNA regulation. We find that fis is not essential for the function of two control systems known to regulate rRNA, growth rate dependent control and stringent control. On the basis of these results, we propose roles for Fis and the upstream activation system in rRNA synthesis.

Base Sequence↗
PubMed1996

Effects of Fis on ribosome synthesis and activity and on rRNA promoter activities in Escherichia coli.

The DNA-bending protein Fis of Escherichia coli is required for efficient initiation of chromosome replication and for activation of the P1 promoters of stable RNA (rRNA and tRNA) genes. Using fis+ and fis- bacterial strains, we have determined ribosome synthesis and activity, rRNA gene dosage, and transcript initiation rates at the rrnB P1 and P2 promoters. Different growth media were used to achieve growth rates between 0.7 and 2.7 doublings/hour for the fis+ strain, and between 0.7 and 2.2 doublings/hour for the fis- strain. In minimal media, the Fis deficiency only reduced the DNA and rrn gene concentration (DNA, oriC copies and rrn genes per protein); in amino acid-supplemented media, it also reduced rRNA synthesis rates per protein. Under all conditions, the ribosome activity (protein synthesis rate/ribosome) remained unchanged by the absence of Fis. In the presence of Fis, the absolute activities of the isolated rrnB P1 and P2 promoters increased from 2 to 88 and 10 to 50 initiations/minute, respectively, with increasing growth-rate. In the absence of Fis, these activities increased from 3 to 70 and 10 to 80 initiations/minute, respectively. Relative to the isolated rrnB P2 promoter, the strength of the rrnB P1 promoter was found to increase with increasing growth rate tenfold (from 0.17 to 1.7) in the presence, but only fivefold (from 0.17 to 0.85) in the absence of Fis. An evaluation of the data leads us to propose estimates of kcat and relative KM values for the two rRNA promoters, and relative values for free RNA polymerase concentrations during growth in different media. The analysis suggests that the reduced strength of P1 promoters of stable RNA genes in the absence of Fis, together with the reduced rrn gene concentration, increases the concentration of free RNA polymerase. In addition, the lower rrn P1 promoter activity in the absence of Fis reduces the probability that the downstream P2 promoter is blocked ("occluded") by a transcription elongation complex originating at the P1 promoter. The increased polymerase concentration and reduced P2 promoter occlusion both help to compensate for the Fis-deficiency, but during growth in rich media when the demand for ribosomes is high, this compensation is insufficient for fis- bacteria to achieve the wild-type level of ribosome synthesis and growth.

Carrier Proteins↗
PubMed1998

DNA microloops and microdomains: a general mechanism for transcription activation by torsional transmission.

Prokaryotic transcriptional activation often involves the formation of DNA microloops upstream of the polymerase binding site. There is substantial evidence that these microloops function to bring activator and polymerase into close spatial proximity. However additional functions are suggested by the ability of certain activators, of which FIS is the best characterised example, to facilitate polymerase binding, promoter opening and polymerase escape. We review here the evidence for the concept that the topology of the microloop formed by such activators is tightly coupled to the structural transitions in DNA mediated by RNA polymerase. In this process, which we term torsional transmission, a major function of the activator is to act as a local topological homeostat. We argue that the same mechanism may also be employed in site-specific DNA inversion.

Carrier Proteins↗
PubMed1999

Conformational changes of the upstream DNA mediated by H-NS and FIS regulate E. coli RrnB P1 promoter activity.

The two proteins FIS and H-NS had previously been shown to regulate ribosomal RNA (rRNA) transcription by interacting with the promoter upstream DNA. FIS is known as an activator whereas H-NS had been demonstrated to function as a repressor. Details of the antagonistic control mechanisms are not yet solved. Here, we have addressed the question how the two proteins cooperate to exert both, positive and negative control of rRNA transcription. By mobility shift experiments and footprinting studies we show that FIS and H-NS binding sites partially overlap but appear to interact with different sites of a curved DNA helix. Although not mutually exclusive, the two proteins compete each other for binding. Both proteins, by changing the DNA curvature, effect circularization reactions of DNA fragments in different ways. Our results imply that binding of the proteins induces alternate DNA conformations with favourable or unfavourable topology for the formation of active transcription complexes. Together the findings presented here help to answer some of the open questions about the concerted molecular mechanism of transcription factors for the regulation of stable RNA synthesis.

Bacterial Proteins↗
PubMed1998

Regulation of transcription initiation at the Escherichia coli nir operon promoter: a new mechanism to account for co-dependence on two transcription factors.

Expression from the Escherichia coli nir promoter is co-dependent on Fnr (a transcription factor triggered by oxygen starvation) and on NarL or NarP (transcription factors triggered by nitrite and nitrate ions). Fnr binds to a single DNA site centred between basepairs 41 and 42 upstream from the nir transcript start, whereas NarL and NarP bind to a site upstream, centred between basepairs 69 and 70. A novel mechanism to account for co-dependence on Fnr and NarL/NarP is suggested from experiments in which the spacing between the DNA sites for Fnr and NarL/NarP was altered. DNA sequence elements located upstream of the NarL/NarP-binding site are the targets for two or more proteins that act to repress Fnr-dependent activation of the nir promoter. This inhibition is counteracted by NarL or NarP. The model has been corroborated by the effects of several deletions and single base substitutions in the nir promoter upstream sequences: these deletions and substitutions prevent the binding of the repressor proteins. One of these repressors has been identified as the Fis protein, that binds to a site located 135-149bp upstream of the nir transcript start: the binding of Fis is suppressed by a single base substitution at position -146. The other repressor protein(s) have yet to be identified, but appear to bind downstream of the DNA site for Fis: binding is suppressed by a single base substitution at position -99.

Bacterial Proteins↗
PubMed1998

Multiple cis-acting sites positively regulate Escherichia coli nrd expression.

Regulation of nrd expression in Escherichia coli by cis-acting elements was found to be more complex than previously reported. At least five upstream sites appear to positively regulate nrd expression including a Fis binding site, a DnaA binding site, an AT-rich region, an inverted repeat and a 10 bp site between the AT-rich region and the inverted repeat. Double mutants defective in these sites indicate that all sites tested act independently when regulating nrd expression. As the decrease in nrd expression in exponentially growing cultures paralleled the decrease observed in DNA synthesis-inhibited cultures for all single and double mutants, we concluded that nrd is regulated by the same mechanism in these physiological states. As mutants unable to induce nrd expression during inhibition of DNA synthesis also fail to exhibit cell cycle-regulated nrd expression, we conclude that cell cycle nrd regulation is controlled by these same sites. Site-directed mutagenesis was used to show that the absence of an increase in nrd expression during DNA inhibition previously observed for deletion of the AT-rich region results from deletion of both the Fis binding site and the AT-rich region.

Artificial Gene Fusion↗
PubMed1999

A DNA architectural protein couples cellular physiology and DNA topology in Escherichia coli.

In Escherichia coli, the transcriptional activity of many promoters is strongly dependent on the negative superhelical density of chromosomal DNA. This, in turn, varies with the growth phase, and is correlated with the overall activity of DNA gyrase, the major topoisomerase involved in the elevation of negative superhelicity. The DNA architectural protein FIS is a regulator of the metabolic reorganization of the cell during early exponential growth phase. We have previously shown that FIS modulates the superhelical density of plasmid DNA in vivo, and on binding reshapes the supercoiled DNA in vitro. Here, we show that, in addition, FIS represses the gyrA and gyrB promoters and reduces DNA gyrase activity. Our results indicate that FIS determines DNA topology both by regulation of topoisomerase activity and, as previously inferred, by directly reshaping DNA. We propose that FIS is involved in coupling cellular physiology to the topology of the bacterial chromosome.

Base Sequence↗
PubMed2000

Suppression of FNR-dependent transcription activation at the Escherichia coli nir promoter by Fis, IHF and H-NS: modulation of transcription initiation by a complex nucleo-protein assembly.

Expression from the Escherichia coli nir promoter is co-dependent on both the FNR protein (an anaerobically triggered transcription activator) and the NarL or NarP proteins (transcription activators triggered by nitrite and nitrate). Under anaerobic conditions, FNR binds to a site centred between positions -41 and -42, activating transcription of the nir operon. In previous work, we showed that this activation is suppressed by the binding of Fis protein, and at least one other protein, to sequence elements located upstream of the nir promoter. We proposed that the binding of NarL or NarP to a site centred between positions -69 and -70 counteracts this suppression, resulting in increased transcription in response to nitrite or nitrate. Here we have further investigated the different proteins that downregulate the nir promoter. We show that the nir promoter is repressed by three DNA binding proteins, Fis, IHF and H-NS. We demonstrate that, in addition to binding to its previously characterized upstream site located at position -142, Fis also binds to a second downstream site located at position +23. A second suppressing factor is IHF, that binds to a site located at position -88. Finally, the nucleoid associated protein, H-NS, preferentially binds to upstream sequences at the nir promoter and represses promoter activity. The association of Fis, IHF and H-NS suggests that nir promoter DNA is sequestrated into a highly ordered nucleo-protein structure that represses FNR-dependent transcription activation. NarL and NarP can relieve both IHF- and Fis-mediated repression, but are unable to counteract H-NS-mediated repression.

Bacterial Proteins↗
PubMed2002

Independent regulation of the divergent Escherichia coli nrfA and acsP1 promoters by a nucleoprotein assembly at a shared regulatory region.

Expression from the Escherichia coli nrfA promoter (pnrfA) is activated by both the FNR protein (an anaerobically triggered transcription activator) and the NarL or NarP proteins (transcription activators triggered by nitrite and nitrate). Under anaerobic conditions, FNR binds to a site centred at position -41.5 at pnrfA and activates transcription. Further activation, induced by the presence of nitrite, results from the binding of NarL and NarP to a site centred at position -74.5. A second promoter (pacsP1), which directs transcription into the adjacent gene encoding acetyl coenzyme A synthetase (acs), is overlapping and divergent to pnrfA. Despite extensive overlap of regulatory elements, pnrfA and pacsP1 are regulated independently. We demonstrate that at least two nucleoid-associated factors bind to the nrfA-acs intergenic region. The Fis protein binds to a site centred at position -15 (in relation to pnrfA transcription), whereas the IHF protein binds to a site centred at position -54. Both Fis and IHF repress in vivo expression from pacsP1, but have smaller repressive effects on expression from pnrfA. Gel retardation assays were used to investigate the pairwise binding of FNR, NarL, Fis and IHF proteins to the nrfA-acs intergenic region. The binding of NarL and IHF is mutually exclusive, whereas all other combinations can bind simultaneously. Experiments in which deletions and point mutations were introduced into the upstream region of pnrfA demonstrated that an additional factor must bind upstream to inhibit FNR-dependent transcription. We conclude that the nrfA-acs intergenic region is folded into an ordered nucleoprotein structure that permits the two divergent promoters to be regulated independently in response to different physiological signals.

Acetate-CoA Ligase↗
PubMed2001

CRP modulates fis transcription by alternate formation of activating and repressing nucleoprotein complexes.

The DNA architectural proteins FIS and CRP are global regulators of transcription in Escherichia coli involved in the adjustment of cellular metabolism to varying growth conditions. We have previously demonstrated that FIS modulates the expression of the crp gene by functioning as its transcriptional repressor. Here we show that in turn, CRP is required to maintain the growth phase pattern of fis expression. We demonstrate the existence of a divergent promoter in the fis regulatory region, which reduces transcription of the fis promoter. In the absence of FIS, CRP activates fis transcription, thereby displacing the polymerase from the divergent promoter, whereas together FIS and CRP synergistically repress fis gene expression. These results provide evidence for a direct cross-talk between global regulators of cellular transcription during the growth phase. This cross-talk is manifested in alternate formation of functional nucleoprotein complexes exerting either activating or repressing effects on transcription.

Base Sequence↗
PubMed2005

Integration of three signals at the Escherichia coli nrf promoter: a role for Fis protein in catabolite repression.

Expression from the Escherichia coli nrf operon promoter is activated by the anaerobically triggered transcription factor, FNR, and by the nitrate/nitrite ion-controlled response regulators, NarL or NarP, but is repressed by the IHF and Fis proteins. Here, we present in vitro studies on the nrf promoter, using permanganate footprinting to measure open complex formation, and DNase I footprinting to monitor binding of the different regulators and the interactions between them. Our results show that open complex formation is completely dependent on FNR and is enhanced by NarL, but is repressed by IHF or Fis. NarL counteracts repression by IHF but is unable to alter repression by Fis. These results suggest mechanisms by which nrf promoter activity is modulated by the different factors. Expression from the nrf promoter is known to be repressed in rich media, especially in the presence of glucose, but the molecular basis of this is not understood. Here, we show that this catabolite repression is relieved by mutations that weaken the DNA site for Fis, improve the DNA site for FNR or improve the promoter -10 or -35 elements. Hence, Fis protein is a major factor responsible for catabolite repression at the nrf promoter, and Fis can override activation by FNR and NarL or NarP.

Base Sequence↗
PubMed1992

Involvement of Fis protein in replication of the Escherichia coli chromosome.

We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in these fis mutant strains following upshift to 44 degrees C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32 degrees C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.

Bacterial Proteins↗
PubMed1992

Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli.

Fis is a small basic DNA-binding protein from Escherichia coli that was identified because of its role in site-specific DNA recombination reactions. Recent evidence indicates that Fis also participates in essential cell processes such as rRNA and tRNA transcription and chromosomal DNA replication. In this report, we show that Fis levels vary dramatically during the course of cell growth and in response to changing environmental conditions. When stationary-phase cells are subcultured into a rich medium, Fis levels increase from less than 100 to over 50,000 copies per cell prior to the first cell division. As cells enter exponential growth, nascent synthesis is largely shut off, and intracellular Fis levels decrease as a function of cell division. Fis synthesis also transiently increases when exponentially growing cells are shifted to a richer medium. The magnitude of the peak of Fis synthesis appears to reflect the extent of the nutritional upshift. fis mRNA levels closely resemble the protein expression pattern, suggesting that regulation occurs largely at the transcriptional level. Two RNA polymerase-binding sites and at least six high-affinity Fis-binding sites are present in the fis promoter region. We show that expression of the fis operon is negatively regulated by Fis in vivo and that purified Fis can prevent stable complex formation by RNA polymerase at the fis promoter in vitro. However, autoregulation only partially accounts for the expression pattern of Fis. We suggest that the fluctuations in Fis levels may serve as an early signal of a nutritional upshift and may be important in the physiological roles Fis plays in the cell.

Amino Acid Sequence↗
PubMed1994

Regulation of the Escherichia coli nrd operon: role of DNA supercoiling.

An in vitro RNA transcription assay was used to investigate the regulation of the expression of the nrd promoter. Using a linear DNA template, we found that Fis protein, which has a positive effect on expression of the nrd promoter in an nrd-lacZ fusion in vivo, had a moderate negative effect in vitro. However, with a supercoiled DNA template as substrate, we found that Fis had a concentration-dependent positive effect on nrd transcription in vitro. This positive effect was not present on two templates that had 35- or 37-bp insertions between the Fis binding site and the nrd promoter. In the absence of Fis protein, a dramatic decrease in transcription was observed in templates with reduced supercoiling generated by the treatment with wheat germ topoisomerase I. Templates with insertions of 35 bp into an HpaII site at -102 or 37 bp into the MnlI site at -33 bp from the start of transcription failed to exhibit the DNA supercoiling sensitivity of the nrd promoter. Analysis of cells containing either of these two nrd-lacZ fusion constructs that has an insertion at the regulatory region by flow cytometry indicated that these two constructs, unlike the parental construct, were not cell cycle regulated.

Base Sequence↗
PubMed1997

Expression of AbrB, a transition state regulator from Bacillus subtilis, is growth phase dependent in a manner resembling that of Fis, the nucleoid binding protein from Escherichia coli.

The transition state regulator AbrB functions as an activator, a repressor, and a preventer of gene expression in Bacillus subtilis. In this paper, we show that expression of abrB is growth phase dependent. Accumulation of abrB transcript is restricted to a short period spanning the transition between the lag and exponential phases of the growth cycle. The level of abrB transcript then falls sharply, and transcript cannot be detected at the mid-exponential period of the growth cycle. The level of AbrB protein is also maximal during early exponential growth but decreases gradually throughout the remainder of the growth cycle. The abrupt reduction of abrB transcript level during the early period of the growth cycle is effected by the phosphorylated form of the response regulator Spo0p3and to a lesser extent by negative autoregulation. The growth cycle-dependent expression of abrB is very similar to that observed for fis in Escherichia coli and in Salmonella typhimurium. Although AbrB and Fis are not homologous proteins, they display extensive similarity in terms of size, DNA binding characteristics, growth cycle-dependent patterns of expression, and their control over the expression of a varied group of operons. We hypothesize therefore that AbrB, like Fis, is a nucleoid binding protein.

Bacillus subtilis↗
PubMed1997

Fis, an accessorial factor for transcriptional activation of the mar (multiple antibiotic resistance) promoter of Escherichia coli in the presence of the activator MarA, SoxS, or Rob.

Transcription of the multiple antibiotic resistance marRAB operon increases when one of the sequence-related activators, MarA, SoxS, or Rob, binds to the "marbox" centered at -61.5 relative to the transcriptional start site. Previous deletion analyses showed that an adjacent upstream "accessory region" was needed to augment the marbox-dependent activation. To analyze the roles of the marbox and accessory regions on mar transcription, thirteen promoters, each with a different 5-bp transversion of the -96 to -32 sequence, were synthesized, fused to lacZ, and assayed for beta-galactosidase production in single-copy lysogens with appropriate genotypes. The accessory region is shown here to be a binding site for Fis centered at -81 and to bind Fis, a small DNA-binding and -bending protein, with a Kd of approximately 5 nM. The binding of MarA to the marbox and that of Fis to its site were independent of each other. MarA, SoxS, and Rob each activated the mar promoter 1.5-to 2-fold when it had a wild-type marbox but Fis was absent. In the presence of MarA, SoxS, or Rob, Fis further enhanced the activity of the promoter twofold provided the promoter was also capable of binding Fis. However, in the absence of MarA, SoxS, or Rob or in the absence of a wild-type marbox, Fis nonspecifically lowered the activity of the mar promoter about 25% whether or not a wild-type Fis site was present. Thus, Fis acts as an accessory transcriptional activator at the mar promoter.

Bacterial Proteins↗