Fis Binding Sites Research Articles

Correlation between binding rate constants and individual information of E. coli Fis binding sites.

Individual protein binding sites on DNA can be measured in bits of information. This information is related to the free energy of binding by the second law of thermodynamics, but binding kinetics appear to be inaccessible from sequence information since the relative contributions of the on- and off-rates to the binding constant, and hence the free energy, are unknown. However, the on-rate could be independent of the sequence since a protein is likely to bind once it is near a site. To test this, we used surface plasmon resonance and electromobility shift assays to determine the kinetics for binding of the Fis protein to a range of naturally occurring binding sites. We observed that the logarithm of the off-rate is indeed proportional to the individual information of the binding sites, as predicted. However, the on-rate is also related to the information, but to a lesser degree. We suggest that the on-rate is mostly determined by DNA bending, which in turn is determined by the sequence information. Finally, we observed a break in the binding curve around zero bits of information. The break is expected from information theory because it represents the coding demarcation between specific and nonspecific binding.

The −35 sequence location and the Fis–sigma factor interface determine σS selectivity of the proP (P2) promoter in Escherichia coli

The P2 promoter of proP, encoding a transporter for proline and glycine betaine in Escherichia coli, is a unique paradigm, where master regulators of different growth stages, Fis and sigma(S) (RpoS), collaborate to achieve promoter activation. It is also the only case described where Fis functions as class II transcriptional activator (centred at -41). Here we show that the degenerate -35 sequence, and the location of the Fis binding site, which forces a suboptimal 16 bp spacing between the -35 and -10 elements, allow only sigma(S) but not sigma(70) to function at proP (P2). Moreover, the interface between Fis and sigma(S) seems better suited to sigma(S), due to a single residue difference between sigma(S) and sigma(70). Nevertheless, Fis can activate RNA polymerase containing sigma(70) at a proP (P2) promoter variant, in which a typical sigma(70)-35 recognition sequence has been introduced at a 17 bp distance from the -10 hexamer. In summary, we elucidate the rules that govern sigma factor selectivity in the presence of a class II activator, provide new insight into transcriptional activation by Fis from this position, and clarify, why the proP (P2) promoter is precisely activated during a short time window of the growth cycle, when Fis and sigma(S) are both present.

Initiation of Heat-Induced Replication Requires DnaA and the L-13-mer of oriC

An upshift of 10 degrees C or more in the growth temperature of an Escherichia coli culture causes induction of extra rounds of chromosome replication. This stress replication initiates at oriC but has functional requirements different from those of cyclic replication. We named this phenomenon heat-induced replication (HIR). Analysis of HIR in bacterial strains that had complete or partial oriC deletions and were suppressed by F integration showed that no sequence outside oriC is used for HIR. Analysis of a number of oriC mutants showed that deletion of the L-13-mer, which makes oriC inactive for cyclic replication, was the only mutation studied that inactivated HIR. The requirement for this sequence was strictly correlated with Benham's theoretical stress-induced DNA duplex destabilization. oriC mutations at DnaA, FIS, or IHF binding sites showed normal HIR activation, but DnaA was required for HIR. We suggest that strand opening for HIR initiation occurs due to heat-induced destabilization of the L-13-mer, and the stable oligomeric DnaA-single-stranded oriC complex might be required only to load the replicative helicase DnaB.

Fis is required for proper regulation of ssaG expression in Salmonella enterica serovar Typhimurium

Salmonella pathogenicity island 2 (SPI2) encodes a type III secretion system (TTSS) necessary for bacterial survival and replication in intracellular environment of host cells. SPI2 genes are transcribed preferentially after Salmonella enters the host cells. Transcriptional regulation of ssaG encoding the component of SPI2–TTSS apparatus was studied in vivo and in vitro. Fis, one of the major components of bacterial nucleoid, activated the stationary phase-specific expression of ssaG when Salmonella was grown in LB media. Gel-shift and footprinting analysis showed Fis bound to four distinct sites of the ssaG promoter region with different affinities. All four Fis-binding sites were required for timely transcription activation of ssaG after Salmonella entered macrophage cells. Gentamicin protection experiments using bacteria grown to stationary phase prior to infection showed that the ability of the fis mutant strain to replicate within the RAW264.7 macrophage cells was lower than the wild type. These observations confirm that Fis plays an important role in regulations of SPI2 as well as SPI1 for an efficient regulation of the virulence genes.

A mutational analysis of the ColE1-encoded cell cycle regulator Rcd confirms its role in plasmid stability

Multimers of multicopy plasmids cause instability. They arise by homologous recombination and accumulate by over-replication in a process known as the dimer catastrophe. Dimers are resolved to monomers by site-specific recombination systems such as Xer- cer of plasmid ColE1. In addition, the Rcd checkpoint hypothesis proposes that a short transcript (Rcd) coded within ColE1 cer delays the division of multimer-containing cells. The crucial observation underpinning the checkpoint hypothesis is that when the Rcd promoter (P cer ) is inactivated by mutation of its invariant T, the plasmid becomes unstable. Recently, we discovered that this mutation also alters a potential Fis binding site in cer. ColE1-like plasmids are less stable in fis mutant hosts and it is conceivable that instability caused by the mutation is due to altered Fis binding, rather than the loss of Rcd expression per se. We have therefore undertaken an independent test of the role of P cer -Rcd in multicopy plasmid stability. We have generated a series of loss-of-function mutants of Rcd and detailed analysis of two of these shows that they cause a level of instability indistinguishable from P cer inactivation. This result is consistent with the predictions of the checkpoint hypothesis and confirms the role of Rcd in plasmid stability.

Buffering of stable RNA promoter activity against DNA relaxation requires a far upstream sequence.

The stable RNA promoters of Escherichia coli are exquisitely sensitive to variations in the superhelical density of DNA. Previously, we have shown that binding of the DNA architectural protein FIS at the upstream activating sequences (UASs) of stable RNA promoters prevents the transcription complexes from inactivation induced by changes in the supercoiling level of DNA. Here, we identify a strong FIS binding site 89 bp upstream of the previously described cluster of FIS binding sites located between positions -64 and -150 in the rrnA P1 UAS. Binding of FIS to this 'far upstream sequence' allows the recruitment of additional FIS molecules to the region. We demonstrate that, upon DNA relaxation, the maintenance of promoter activity requires, in addition to UAS, the presence of the far upstream sequence. The far upstream sequence shows no effect in the absence of an intact cluster. This requirement for the integrity of the region encompassing the far upstream sequence and the UAS cluster is correlated with the in vitro modulation of binding of FIS to UAS and interaction of RNA polymerase with the UP element and the region around the transcriptional start point. Our results suggest that, at the rrnA P1 promoter, the entire region comprising the UAS and the far upstream sequence is involved in the assembly of the transcription initiation complex. We propose that the extensive engagement of upstream DNA in this nucleoprotein complex locally compensates for the lack of torsional strain in relaxed DNA, thus increasing the resistance of the promoter to global DNA relaxation.

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.

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.

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 crp transcription by oscillation between distinct nucleoprotein complexes.

FIS belongs to the group of small abundant DNA-binding proteins of Escherichia coli. We recently demonstrated that, in vivo, FIS regulates the expression of several genes needed for catabolism of sugars and nucleic acids, a majority of which are also transcriptionally regulated by cAMP-cAMP-receptor protein (CRP) complex. Here we provide evidence that FIS represses transcription of the crp gene both in vivo and in vitro. Employing crp promoter-lacZ fusions, we demonstrate that both FIS and cAMP-CRP are required to keep the crp promoter in a repressed state. We have identified in the crp promoter other transcription initiation sites which are located 73, 79 and 80 bp downstream from the previously mapped start site. Two CRP- and several FIS-binding sites with different affinities are located in the crp promoter region, one of them overlapping the downstream transcription initiation sites. We show that initiation of transcription at the crp promoter is affected by the composition of nucleoprotein complexes resulting from the outcome of competition between proteins for overlapping binding sites. Our results suggest that the control of crp transcription is achieved by oscillation in the composition of these regulatory nucleoprotein complexes in response to the physiological state of the cell.

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.

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.

FIS activates sequential steps during transcription initiation at a stable RNA promoter.

FIS (factor for inversion stimulation) is a small dimeric DNA-bending protein which both stimulates DNA inversion and activates transcription at stable RNA promoters in Escherichia coli. Both these processes involve the initial formation of a complex nucleoprotein assembly followed by local DNA untwisting at a specific site. We have demonstrated previously that at the tyrT promoter three FIS dimers are required to form a nucleoprotein complex with RNA polymerase. We now show that this complex is structurally dynamic and that FIS, uniquely for a prokaryotic transcriptional activator, facilitates sequential steps in the initiation process, enabling efficient polymerase recruitment, untwisting of DNA at the transcription startpoint and finally the escape of polymerase from the promoter. Activation of all these steps requires that the three FIS dimers bind in helical register. We suggest that FIS acts by stabilizing a DNA microloop whose topology is coupled to the local topological transitions generated during the initiation of transcription.