Fis Binding Research Articles

Promoter protection by a transcription factor acting as a local topological homeostat.

Binding of the Escherichia coli global transcription factor FIS to the upstream activating sequence (UAS) of stable RNA promoters activates transcription on the outgrowth of cells from stationary phase. Paradoxically, while these promoters require negative supercoiling of DNA for optimal activity, FIS counteracts the increase of negative superhelical density by DNA gyrase. We demonstrate that binding of FIS at the UAS protects the rrnA P1 promoter from inactivation at suboptimal superhelical densities. This effect is correlated with FIS-dependent constraint of writhe and facilitated untwisting of promoter DNA. We infer that FIS maintains stable RNA transcription by stabilizing local writhe in the UAS. These results suggest a novel mechanism of transcriptional regulation by a transcription factor acting as a local topological homeostat.

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.

An architectural role of the Escherichia coli chromatin protein FIS in organising DNA.

The Escherichia coli chromatin protein FIS modulates the topology of DNA in a growth phase-dependent manner. In this study we have investigated the global effect of FIS binding on DNA architecture in vitro. We show that in supercoiled DNA molecules FIS binds at multiple sites in a non-random fashion and increases DNA branching. This global DNA reshaping effect is independent of the helical phasing of FIS binding sites. We propose, in addition to the previously inferred stabilisation of tightly bent DNA microloops in the upstream regions of certain promoters, that FIS may perform the distinct architectural function of organising branched plectonemes in the E.coli nucleoid.

Contributions of UP elements and the transcription factor FIS to expression from the seven rrn P1 promoters in Escherichia coli.

The high activity of the rrnB P1 promoter in Escherichia coli results from a cis-acting DNA sequence, the UP element, and a trans-acting transcription factor, FIS. In this study, we examine the effects of FIS and the UP element at the other six rrn P1 promoters. We find that UP elements are present at all of the rrn P1 promoters, but they make different relative contributions to promoter activity. Similarly, FIS binds upstream of, and activates, all seven rrn P1 promoters but to different extents. The total number of FIS binding sites, as well as their positions relative to the transcription start site, differ at each rrn P1 promoter. Surprisingly, the FIS sites upstream of site I play a much larger role in transcription from most rrn P1 promoters compared to rrnB P1. Our studies indicate that the overall activities of the seven rrn P1 promoters are similar, and the same contributors are responsible for these high activities, but these inputs make different relative contributions and may act through slightly different mechanisms at each promoter. These studies have implications for the control of gene expression of unlinked multigene families.

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.

The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome.

We have developed a simple three-step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild-type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.

Antagonistic control of the Escherichia coli bgl promoter by FIS and CAP in vitro.

The wild-type Escherichia coli bgl promoter is silent in vivo but active in vitro. Silencing in vivo is directed by silencer sequences that flank the promoter, and requires nucleoid-associated protein H-NS and other unidentified cellular factors. Here we show that the DNA bending protein FIS is a repressor of the bgl promoter. Two FIS binding sites, centred at positions -52 and -27, overlap the CAP binding site and the -35 box respectively. FIS efficiently competes with CAP for binding to the wild-type promoter. However, FIS does not prevent binding of RNA polymerase. It interferes with the formation of a heparin-resistant complex and represses transcription initiation up to 40-fold. The presence of CAP has very little effect on the FIS-mediated repression of the wild-type bgl promoter in vitro. However, when a bgl promoter allele was tested that carries an improved CAP binding site (which leads to activation in vivo) CAP effectively counteracted repression by FIS in vitro. These results suggest that FIS contributes to silencing of the wild-type bgl promoter in vivo, presumably in the early exponential phase when FIS is predominantly expressed.

Drunken-cell footprints: nuclease treatment of ethanol-permeabilized bacteria reveals an initiation-like nucleoprotein complex in stationary phase replication origins.

The nucleoprotein complex formed on oriC, the Escherichia coli replication origin, is dynamic. During the cell cycle, high levels of the initiator DnaA and a bending protein, IHF, bind to oriC at the time of initiation of DNA replication, while binding of Fis, another bending protein, is reduced. In order to probe the structure of nucleoprotein complexes at oriC in more detail, we have developed an in situ footprinting method, termed drunken-cell footprinting, that allows enzymatic DNA modifying reagents access to intracellular nucleoprotein complexes in E.coli, after a brief exposure to ethanol. With this method, we observed in situ binding of Fis to oriC in exponentially growing cells, and binding of IHF to oriC in stationary cells, using DNase I and Bst NI endonuclease, respectively. Increased binding of DnaA to oriC in stationary phase was also noted. Because binding of DnaA and IHF results in unwinding of oriC in vitro, P1 endonuclease was used to probe for intracellular unwinding of oriC. P1 cleavage sites, localized within the 13mer unwinding region of oriC ', were dramatically enhanced in stationary phase on wild-type origins, but not on mutant versions of oriC unable to unwind. These observations suggest that most oriC copies become unwound during stationary phase, forming an initiation-like nucleoprotein complex.

Regulation of adhE (encoding ethanol oxidoreductase) by the Fis protein in Escherichia coli.

The adhE gene of Escherichia coli encodes a multifunctional ethanol oxidoreductase whose expression is 10-fold higher under anaerobic than aerobic conditions. Transcription of the gene is under the negative control of the Cra (catabolite repressor-activator) protein, whereas translation of the adhE mRNA requires processing by RNase III. In this report, we show that the expression of adhE also depends on the Fis (factor for inversion stimulation) protein. A strain bearing a fis::kan null allele failed to grow anaerobically on glucose solely because of inadequate adhE transcription. However, fis expression itself is not under redox control. Sequence inspection of the adhE promoter revealed three potential Fis binding sites. Electrophoretic mobility shift analysis, using purified Fis protein and adhE promoter DNA, showed three different complexes.

Activation of Escherichia coli leuV transcription by FIS.

The transcription factor FIS has been implicated in the regulation of several stable RNA promoters, including that for the major tRNALeu species in Escherichia coli, tRNA1Leu. However, no evidence for direct involvement of FIS in tRNA1Leu expression has been reported. We show here that FIS binds to a site upstream of the leuV promoter (centered at -71) and that it directly stimulates leuV transcription in vitro. A mutation in the FIS binding site reduces transcription from a leuV promoter in strains containing FIS but has no effect on transcription in strains lacking FIS, indicating that FIS contributes to leuV expression in vivo. We also find that RNA polymerase forms an unusual heparin-sensitive complex with the leuV promoter, having a downstream protection boundary of approximately -7, and that the first two nucleotides of the transcript, GTP and UTP, are required for formation of a heparin-stable complex that extends downstream of the transcription start site. These studies have implications for the regulation of leuV transcription.

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.

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.

Information analysis of Fis binding sites.

Originally discovered in the bacteriophage Mu DNA inversion system gin, Fis (Factor for Inversion Stimulation) regulates many genetic systems. To determine the base frequency conservation required for Fis to locate its binding sites, we collected a set of 60 experimentally defined wild-type Fis DNA binding sequences. The sequence logo for Fis binding sites showed the significance and likely kinds of base contacts, and these are consistent with available experimental data. Scanning with an information theory based weight matrix within fis, nrd, tgt/sec and gin revealed Fis sites not previously identified, but for which there are published footprinting and biochemical data. DNA mobility shift experiments showed that a site predicted to be 11 bases from the proximal Salmonella typhimurium hin site and a site predicted to be 7 bases from the proximal P1 cin site are bound by Fis in vitro. Two predicted sites separated by 11 bp found within the nrd promoter region, and one in the tgt/sec promoter, were also confirmed by gel shift analysis. A sequence in aldB previously reported to be a Fis site, for which information theory predicts no site, did not shift. These results demonstrate that information analysis is useful for predicting Fis DNA binding.

Information Content of Individual Genetic Sequences

Related genetic sequences having a common function can be described by Shannon's information measure and depicted graphically by a sequence logo. Though useful for many purposes, sequence logos only show the average sequence conservation, and inferring the conservation for individual sequences is difficult. This limitation is overcome by the individual information (Ri) technique described here. The method begins by generating a weight matrix from the frequencies of each nucleotide or amino acid at each position of the aligned sequences. This matrix is then applied to the sequences themselves to determine the sequence conservation of each individual sequence. The matrix is unique because the average of these assignments is the total sequence conservation, ad there is only one way to construct such a matrix. For binding sites on polynucleotides, the weight matrix has a natural cut off that distinguishes functional sequences from other sequences.Rivalues are on an absolute scale measured in bits of information so the conservation of different biological functions can be compared with one another. The matrix can be used to rank-order the sequences, to search for new sequences, to compare sequences to other quantitative data such as binding energy or distance between binding sites, to distinguish mutations from polymorphisms, to design sequences of a given strength, and to detect errors in databases. TheRimethod has been used to identify previously undescribed but experimentally verified DNA binding sites. The individual information distribution was determined forE. coliribosome binding sites, bacterial Fis binding sites, and human donor and acceptor splice junctions, among others. The distributions demonstrate clearly that the consensus sequence is highly unusual, and hence is a poor method to describe naturally occurring binding sites.

Stimulation of DNA inversion by FIS: evidence for enhancer-independent contacts with the Gin-gix complex.

Efficient DNA inversion catalysed by the invertase Gin requires the cis-acting recombinational enhancer and the Escherichia coliFIS protein. Binding of FIS bends the enhancer DNA and, on a negatively supercoiled DNA inversion substrate, facilitates the formation of a synaptic complex with specific topology. Previous studies have indicated that FIS-independent Gin mutants can be isolated which have lost the topological constraints imposed on the inversion reaction yet remain sensitive to the stimulatory effect of FIS. Whether the effect of FIS is purely architectural, or whether in addition direct protein contacts between Gin and FIS are required for efficient catalysis has remained an unresolved question. Here we show that FIS mutants impaired in DNA binding are capable of either positively or negatively affecting the inversion reaction both in vivo and in vitro. We further demonstrate that the mutant protein FIS K25E/V66A/M67T dramatically enhances the cleavage of recombination sites by FIS-independent Gin in an enhancer-independent manner. Our observations suggest that FIS plays a dual role in the inversion reaction and stimulates both the assembly of the synaptic complex as well as DNA strand cleavage.

Deletion analysis of the fis promoter region in Escherichia coli: antagonistic effects of integration host factor and Fis.

Fis is a small DNA-binding and -bending protein in Escherichia coli that is involved in several different biological processes, including stimulation of specialized DNA recombination events and regulation of gene expression. fis protein and mRNA levels rapidly increase during early logarithmic growth phase in response to a nutritional upshift but become virtually undetectable during late logarithmic and stationary phases. We present evidence that the growth phase-dependent fis expression pattern is not determined by changes in mRNA stability, arguing in favor of regulation at the level of transcription. DNA deletion analysis of the fis promoter (fis P) region indicated that DNA sequences from -166 to -81, -36 to -26, and +107 to +366 relative to the transcription start site are required for maximum expression. A DNA sequence resembling the integration host factor (IHF) binding site centered approximately at -114 showed DNase I cleavage protection by IHF. In ihf cells, maximum cellular levels of fis mRNA were decreased more than 3-fold and transcription from fis P on a plasmid was decreased about 3.8-fold compared to those in cells expressing wild-type IHF. In addition, a mutation in the ihf binding site resulted in a 76 and 61% reduction in transcription from fis P on a plasmid in the presence or absence of Fis, respectively. Insertions of 5 or 10 bp between this ihf site and fis P suggest that IHF functions in a position-dependent manner. We conclude that IHF plays a role in stimulating transcription from fis P by interacting with a site centered approximately at -114 relative to the start of transcription. We also showed that although the fis P region contains six Fis binding sites, Fis site II (centered at -42) played a predominant role in autoregulation, Fis sites I and III (centered at +26 and -83, respectively) seemingly played smaller roles, and no role in negative autoregulation could be attributed to Fis sites IV, V, and VI (located upstream of site III). The fis P region from -36 to +7, which is not directly regulated by either IHF or Fis, retained the characteristic fis regulation pattern in response to a nutritional upshift.

Location, degree, and direction of DNA bending associated with the Hin recombinational enhancer sequence and Fis-enhancer complex.

The Fis protein of Escherichia coli and Salmonella typhimurium stimulates several site-specific DNA recombination reactions, as well as transcription of a number of genes. Fis binds to a 15-bp core recognition sequence and induces DNA bending. Mutations in Fis which alter its ability to bend DNA have been shown to reduce the stimulatory activity of Fis in both site-specific recombination and transcription systems. To examine the role of DNA bending in the activity of the Fis-recombinational enhancer complex in Hin-mediated site-specific DNA inversion, we have determined the locations, degrees, and directions of DNA bends associated with the recombinational enhancer and the Fis-enhancer complex. Circular-permutation assays demonstrated that a sequence-directed DNA bend is associated with the Fis binding sites in the proximal and distal domains of the recombinational enhancer. Binding of Fis to its core recognition sequence significantly increases the degree of DNA bending associated with the proximal and distal domains. The degree of DNA bending induced by Fis binding depended on the DNA sequences flanking the core Fis binding site, with angles ranging from 42 to 69 degrees. Phasing analyses indicate that both the sequence-directed and the Fis-induced DNA bends associated with the proximal and distal domains face the minor groove of the DNA helix at the center of the Fis binding site. The positions and directions of DNA bends associated with the Fis-recombinational complex support a direct role for Fis-induced DNA bending in assembly of the active invertasome.

Induction and repair of cyclobutane pyrimidine dimers in the Escherichia coli tRNA gene tyrT:Fis protein affects dimer induction in the control region and suppresses preferential repair in the coding region of the transcribed strand, except in a short region near the transcription start site

We analysed induction and repair of UV induced pyrimidine dimers in the Escherichia coli tRNA gene tyrT. In wild-type (WT) log or stationary phase different patterns of induction occurred in the three Fis binding sites and the core promoter −35 sequence of the control region: this was absent in fis − cells. In stationary WT cells, slow, similar rates of repair occurred throughout the non-transcribed strand (NTS). Faster repair occurred in the NTS control region in WT log phase. NTS repair in fis − cells was similar, except the control region differed less between phases. Heterogeneous repair occurred along the transcribed strand (TS). In the control region repair was faster than in the NTS. Repair in the TS coding region changed between growth phases or if repair took place in different media. When irradiated log phase WT cells were in rich medium, two TS domains were evident: a fast-repaired domain within 31 nucleotides from the transcription start site; and a more slowly repaired domain composed of the rest of the TS. A sharp gradient existed in the small domain with very fast repair at the beginning and diminished repair towards the end. Fast transcription coupled repair (TCR) in the small domain was absent in the TS large domain, where repair was similar to the NTS and to the entire TS in mfd − cells. In similarly treated stationary phase WT cells, TCR occurred in the large domain. Depletion of Fis reinstates TCR to a lesser extent, whilst a substitution of five nucleotides at the Fis binding sites in the upstream activating sequence reinstates TCR. Reinstatement of TCR was also achieved by incubating irradiated WT cells in minimal salt medium without the required amino acid. Our results suggest that Fis indirectly suppresses preferential repair in the TS large domain by stimulating transcription.

Sequence Analysis of Fis Binding Sites Obtained by in Vitro Selection

To study the specificity of the FIS protein binding to DNA, we have isolated new binding sites by in vitro selection, analyzed their binding and bending by gel electro-phoresis and compared the results to predictions based on sequence analysis of the known natural sites.

Molecular anatomy of a transcription activation patch: FIS-RNA polymerase interactions at the Escherichia coli rrnB P1 promoter.

FIS, a site-specific DNA binding and bending protein, is a global regulator of gene expression in Escherichia coli. The ribosomal RNA promoter rrnB P1 is activated 3- to 7-fold in vivo by a FIS dimer that binds a DNA site immediately upstream of the DNA binding site for the C-terminal domain (CTD) of the alpha subunit of RNA polymerase (RNAP). In this report, we identify several FIS side chains important specifically for activation of transcription at rrnB P1. These side chains map to positions 68, 71 and 74, in and flanking a surface-exposed loop adjacent to the helix-turn-helix DNA binding motif of the protein. We also present evidence suggesting that FIS activates transcription at rrnB P1 by interacting with the RNAP alphaCTD. Our results suggest a model for FIS-mediated activation of transcription at rrnB P1 that involves interactions between FIS and the RNAP alphaCTD near the DNA surface. Although FIS and the transcription activator protein CAP have little structural similarity, they both bend DNA, use a similarly disposed activation loop and target the same region of the RNAP alphaCTD, suggesting that this is a common architecture at bacterial promoters.