Fis Binding Research Articles

Fis binding in the dnaA operon promoter region.

The region between the rpmH and dnaA genes contains five promoters that divergently express the ribosomal protein L34 and the proteins of the dnaA operon, including DnaA, the beta clamp of DNA polymerase III holoenzyme, and RecF. The DNA-binding protein Fis was shown by the band shift assay to bind near the rpmHp2 and dnaAp2 promoters and by DNase I footprinting to bind to a single site in the dnaAp2 promoter overlapping the -35 and spacer sequences. There were no observable differences in Fis affinity or the angle of bending induced by Fis between methylated and unmethylated DNA fragments containing the Fis binding site in the dnaAp2 promoter. Fis directly or indirectly represses the expression of DnaA protein and the beta clamp of DNA polymerase III. A fis null mutant containing a dnaA-lacZ in-frame fusion had twofold greater beta-galactosidase activity than a fis wild-type strain, and induced expression of Fis eliminated the increase in activity of the fusion protein. A two- to threefold increase in the levels of DnaA and beta clamp proteins was found in a fis null mutant by immunoblot gel analysis.

The Escherichia coli Fis protein prevents initiation of DNA replication from oriC in vitro

Fis protein participates in the normal control of chromosomal replication in Escherichia coli. However, the mechanism by which it executes its effect is largely unknown. We demonstrate an inhibitory influence of purified Fis protein on replication from oriC in vitro. Fis inhibits DNA synthesis equally well in replication systems either dependent upon or independent of RNA polymerase, even when the latter is stimulated by the presence of HU or IHF. The extent of inhibition by Fis is modulated by the concentrations of DnaA protein and RNA polymerase; the more limiting the amounts of these, the more severe the inhibition by Fis. Thus, the level of inhibition seems to depend on the ease with which the open complex can be formed. Fis-mediated inhibition of DNA replication does not depend on a functional primary Fis binding site between DnaA boxes R2 and R3 in oriC, as mutations that cause reduced binding of Fis to this site do not affect the degree of inhibition. The data presented suggest that Fis prevents formation of an initiation-proficient structure at oriC by forming an alternative, initiation-preventive complex. This indicates a negative role for Fis in the regulation of replication initiation.

Identification of new Fis binding sites by DNA scission with Fis-1,10-phenanthroline-copper(I) chimeras.

The chimeric nuclease Fis-OP has been used to identify novel Fis binding sites. Tethering the chemical nuclease OP-Cu+ to position 73 of the protein with a newly developed longer acetyl-beta-alanylamino spacer has facilitated the localization of two high-affinity Fis binding sequences in a 3 kb pUC19 plasmid. The shorter acetamido linker has allowed the chimeric nuclease to locate two strong Fis binding sites in the 50 kb phage lambda genome. All four sites reside in biologically interesting loci and have been confirmed by gel-retardation and DNase I footprint analyses. A newly discovered site resides in the lac operon of Escherichia coli. The binding of Fis to this site may antagonize repression by the LacI repressor. These studies demonstrate the feasibility of applying chimeric chemical nucleases to the task of identifying functional protein binding sites of biological interest within genomes without any assumption about their sequence preference.

Fis activates the RpoS-dependent stationary-phase expression of proP in Escherichia coli.

Fis is a general nucleoid-associated protein in Escherichia coli whose expression is highly regulated with respect to growth conditions. A random collection of transposon-induced lac fusions was screened for those which give increased expression in the presence of Fis in order to isolate a ProP-LacZ protein fusion. We find that proP, which encodes a low-affinity transporter of the important osmoprotectants proline and glycine betaine, is transcribed from two promoters. proP1 is transiently induced upon subculture and is upregulated by increases in medium osmolarity. As cells enter stationary phase, a second promoter, proP2, is strongly induced. This promoter can also be induced by high medium osmolarity in exponential phase. The activity of proP2 depends on Fis and the stationary-phase sigma factor sigmas. In the presence of Fis, proP2 expression is increased over 50-fold, as judged by the LacZ activity of cells carrying the proP-lacZ fusion as well as by direct RNA analysis, making this the most strongly activated promoter by Fis that has been described. Two Fis binding sites centered at positions -41 (site I) and -81 (site II) with respect to the transcription initiation site of P2 have been defined by DNase I footprinting. Mutations in site I largely abolish stationary-phase activation, while mutations at site II have a minor effect, suggesting that direct binding of Fis to site I is important for Fis-mediated activation of this promoter. In addition to Fis and sigmas, sequences located over 108 bp upstream of the proP2 transcription initiation site are required for efficient expression.

AldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp

Escherichia coli aldB was identified as a gene that is negatively regulated by Fis but positively regulated by RpoS. The complete DNA sequence determined in this study indicates that aldB encodes a 56.3-kDa protein which shares a high degree of homology with an acetaldehyde dehydrogenase encoded by acoD of Alcaligenes eutrophus and an aldehyde dehydrogenase encoded by aldA of Vibrio cholerae and significant homology with a group of other aldehyde dehydrogenases from prokaryotes and eukaryotes. Expression of aldB is maximally induced during the transition from exponential phase to stationary phase. Its message levels are elevated three- to fourfold by a fis mutation and abolished by an rpoS mutation. In addition, the expression of an aldB-lacZ fusion was decreased about 20-fold in the absence of crp. DNase I footprinting analysis showed that five Fis binding sites and one Crp binding site are located within the aldB promoter region, suggesting that Fis and Crp are acting directly to control aldB transcription. AldB expression is induced by ethanol, but in contrast to that of most of the RpoS-dependent genes, the expression of aldB is not altered by an increase in medium osmolarity.

FIS and RNA polymerase holoenzyme form a specific nucleoprotein complex at a stable RNA promoter.

The Escherichia coli DNA binding protein FIS activates stable RNA promoters during outgrowth of cells from stationary phase. The upstream activating sequences (UASs) of these promoters contain three highly conserved FIS binding sites positioned in helical register. Neither the apparent requirement for three sites nor the mechanism of FIS-mediated activation has been established. We demonstrate here that on saturation of its three binding sites in the UAS, FIS forms a specific nucleoprotein complex which 'traps' RNA polymerase (RNAP) at the promoter of the tyrT operon. This effect is abolished by a change in helical phasing between FIS sites II and III, which impaires cooperative interactions between DNA-bound FIS dimers. The sigma 70 subunit of RNAP stimulates the formation of higher order FIS complexes, a property that is indicative of protein-protein interactions. We propose that after initiation of transcription, the released sigma 70 subunit may be recaptured by the FIS nucleoprotein 'trap' and recycled in successive rounds of holoenzyme assembly. Such a mechanism could overcome transient limitations on the availability of sigma 70 or core polymerase after a prolonged stationary phase.

The Transcriptional Activator Protein FIS: DNA Interactions and Cooperative Interactions with RNA Polymerase at the Escherichia coli rrnBP1 Promoter

The E. coli rrnBP1 promoter oves its stgrength, in part, to the transcriptional activator protein FIS, FIS binds to three sites upsteam of the RNA polymerase (RNAP) binding site and increases transcription in vivofour to ten-fold. In this report, hydroxyl radical and DMS footprinting analyses show that FIS binds to its three sites along one side of the DNA helix, and that FIS bound at the promoter-proximal site (site I) and RNAP bound at the promoter are in close proooooximity. The binding of FIS at site I and RNAP at the promoter are mutually cooperative. These observations support a model for direct interaction between the FIS protein bound at site I and RNAP in transcription activation at rrnBP1. We also find that FIS does not bind cooperatively to its three sites upsteam of rrnBP1, and that the relatively small activation associated with FIS bound at sites II and III does not result indirectly by facilitation of binding of FIS to site I.

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.

Structure of the Escherichia coli Fis-DNA complex probed by protein conjugated with 1,10-phenanthroline copper(I) complex.

The Escherichia coli Fis (factor for inversion stimulation) protein functions in many diverse biological systems including recombination, transcription, and DNA replication. Although Fis is a site-specific DNA-binding protein, it lacks a well-defined consensus recognition sequence. The electrophoretic mobility of Fis-DNA complexes, along with considerations of the Fis crystal structure, indicates that significant deformation of DNA occurs upon Fis binding. To investigate the structure of Fis-DNA complexes, the chemical nuclease 1,10-phenanthroline-copper complex (OP-Cu) has been linked to four specific sites within the Fis DNA-binding domain. Two of these Fis-OP derivatives were active in cleaving DNA. The scission patterns obtained on four different Fis binding sites indicate that Fis positions itself on these highly divergent DNA sequences in a very similar fashion. The patterns of cleavage of a derivative at Asn-98 generally support a model of a Fis-DNA complex that contains specific bends within the core-recognition sequence. Data from a second Fis-OP derivative at Asn-73 provides evidence for greater wrapping of flanking DNA around the sides of the Fis protein than was previously postulated. The cleavage efficiency of flanking segments varies, suggesting that the extent of DNA wrapping is sequence dependent. Specific amino acids on Fis are implicated in promoting this DNA wrapping.

Interaction of Fis protein with DNA: Bending and specificity of binding

The Escherichia coli Fis protein is a dimeric DNA-binding protein whose specific binding sites share a weak consensus sequence. Use of the gel retardation technique indicates that binding of Fis on a linear DNA fragment leads to the formation of a ladder of defined retarded complexes, independently of the presence of a specific site. This non-specific binding of Fis is consistent with a model where equivalent low-affinity sites on a given fragment would be bound randomly and independently of each other by consecutive Fis dimers. Evidence is presented that non-specific binding of Fis can, however, induce an apparent site-specific conformational change in the DNA. This observation is discussed in terms of a model in which each Fis:DNA complex detected in gel retardation experiments actually represents a dynamic equilibrium of a fixed number of Fis dimers distributed on the fragment.

Characterization of the bacteriophage lambda excisionase (Xis) protein: the C-terminus is required for Xis-integrase cooperativity but not for DNA binding.

We have performed a mutational analysis of the xis gene of bacteriophage lambda. The Xis protein is 72 amino acids in length and required for excisive recombination. Twenty-six mutants of Xis were isolated that were impaired or deficient in lambda excision. Mutant proteins that contained amino acid substitutions in the N-terminal 49 amino acids of Xis were defective in excisive recombination and were unable to bind DNA. In contrast, one mutant protein containing a leucine to proline substitution at position 60 and two truncated proteins containing either the N-terminal 53 or 64 amino acids continued to bind lambda DNA, interact cooperatively with FIS and promote excision. However, these three mutants were unable to bind DNA cooperatively with Int. Cooperativity between wild-type Xis and Int required the presence of FIS, but not the Int core-type binding sites. This study shows that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision.

Analysis of the Fis-dependent and Fis-independent transcription activation mechanisms of the Escherichia coli ribosomal RNA P1 promoter

The role of the curved DNA sequence upstream to the Escherichia coli ribosomal RNA P1 promoter in transcription activation was studied. This sequence region had been shown to activate transcription from P1 in vivo and in vitro and to harbor binding sites for the trans-activating protein Fis. We have constructed a series of linker scanning mutants spanning the region -104 to -47, relative to the transcription start site. DNA fragments carrying the mutations show altered gel electrophoretic mobilities, consistent with reduced DNA bending angles compared to the wild-type sequence. Using gel retardation assays, qualitative as well as quantitative differences in the binding of the trans-activating protein Fis to the mutant DNA fragments could be observed. The effects of the mutations on rrnB P1 promoter activation were studied in vivo in fis+ and fis- backgrounds. A reduction in the promoter strength for some of the linker mutants correlates with altered Fis binding to two of the known Fis binding sites. Shifting the Fis binding region by half a helical turn, relative to the promoter core sequence, abolishes Fis-mediated activation almost totally, whereas activation is partly restored by a shift of a complete helical turn. For one mutant, which does not show alterations in Fis binding, a decrease in the promoter strength was observed in a fis- strain. From the results, we conclude that two upstream activating mechanisms, one Fis-dependent and one Fis-independent, influence the rrnB P1 promoter strength. Sequence determinants for the Fis-independent mechanism are closer to the promoter core region than the Fis binding sites. In addition, the study demonstrates that both the helical geometry and the absolute distance of the UAS region relative to the promoter are crucial for transcription activation.

Both fis-dependent and factor-independent upstream activation of the rrnB P1 promoter are face of the helix dependent.

Transcription from the Escherichia coli rrnB P1 promoter is increased by a cis-acting sequence which extends upstream of the -35 hexamer to about -150 with respect to the transcription initiation site, the Upstream Activation Region (UAR). Activation by the UAR involves two components: (1) a trans-acting protein, Fis, which binds to three sites in the UAR between -60 and -150, and (2) the UAR sequences themselves which affect RNA polymerase (RNAP) activity independent of other proteins. We refer to the latter as Factor-Independent Activation (FIA). In addition to its interactions with the -10 and -35 hexamers typical of E. coli promoters, RNAP makes contacts to the -53 region of rrnB P1, which may be related to the FIA effect. We constructed a series of insertion mutants containing integral and non-integral numbers of helical turns at position -46, between the Fis binding sites and the -35 region, and the resulting promoter activities were measured in vitro and in vivo. The data suggest that both Fis-dependent and factor-independent activation are face of the helix dependent: the Fis binding site and the sequences responsible for factor-independent activation must be correctly oriented relative to RNA polymerase in order to activate transcription. These results, in conjunction with other evidence, support a model for the involvement of direct Fis-RNAP interactions in upstream activation. We also demonstrate that RNAP interacts with the -53 region of the rrnB P1 UAR even when these sequences are displaced upstream of the RNAP binding site, and that these interactions correlate with factor-independent activation.

A genetic analysis of Xis and FIS interactions with their binding sites in bacteriophage lambda

The bacteriophage P22-based challenge-phage system was used to study the binding of Xis and FIS to their sites in attP of bacteriophage lambda. Challenge phages were constructed that contained the X1, X2, and F sites within the P22 Pant promoter, which is required for expression of antirepressor. If Xis and FIS bind to these sites in vivo, they repress transcription from Pant, allowing lysogenization to occur. Challenge phages carrying the XIX2F region in either orientation exhibited lysogenization dependent on both Xis and FIS. Neither Xis nor FIS was capable of functioning by itself as an efficient repressor in this system. This was the first time challenge phages have been constructed that require two different proteins bound simultaneously to act as a repressor. Mutations in the X1, X2, and F sites that inhibit Xis and FIS from binding were isolated by selecting mutant phages that still expressed antirepressor synthesis in the presence of Xis and FIS. DNA sequence analysis of the mutants revealed 38 unique mutations, including single-base-pair substitutions, multiple-base-pair changes, deletions, and insertions throughout the entire X1, X2, and F regions. Some of the mutations verified the importance of certain bases within the proposed consensus sequences for Xis and FIS, while others provided evidence that the DNA sequence outside of the proposed binding sites may affect the binding of the individual proteins or the cooperativity between them.

Multiple effects of Fis on integration and the control of lysogeny in phage lambda

Fis is a small, basic, site-specific DNA-binding protein present in Escherichia coli. A Fis-binding site (F) has been previously identified in the attP recombination site of phage lambda (J. F. Thompson, L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy, Cell 50:901-908, 1987). The present study demonstrates that in the absence of the phage-encoded Xis protein, the binding of Fis to F can stimulate integrative recombination and therefore increase the frequency of lambda lysogeny in vivo. Additionally, Fis exerts a stimulatory effect on both integration and lysogeny that is independent of binding to the attP F site. Maintenance of the lysogenic state also appears to be enhanced in the presence of Fis, as shown by the increased sensitivity of lambda prophages encoding temperature-sensitive repressors to partial thermoinduction in a fis mutant. In the presence of Xis, however, Fis binding to F interferes with integration by stimulating excision, the competing back-reaction. Since Fis stimulates both excision and integration, depending on the presence or absence of Xis, respectively, we conclude that Xis binding to X1 is the key determinant directing the formation of an excisive complex.

Efficient excision of phage lambda from the Escherichia coli chromosome requires the Fis protein

The Escherichia coli protein Fis has been shown to bind a single site in the recombination region of phage lambda and to stimulate excisive recombination in vitro (J. F. Thompson, L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy, Cell 50:901-908, 1987). We demonstrate that mutant strains deficient in fis expression show dramatically reduced rates of lambda excision in vivo. Phage yields after induction of a stable lysogen are reduced more than 200-fold in fis cells. The defect observed in phage yield is not due to inefficient phage replication or lytic growth. Direct examination of excisive recombination products reveals a severe defect in the rate of recombination in the absence of Fis. The excision defect observed in fis cells can be fully reproduced in fis+ cells by using phages that lack the Fis binding site on attR, indicating that the entire stimulatory effect of Fis on excisive recombination is due to binding at that site.

The FIS protein binds and bends the origin of chromosomal DNA replication,oriC, ofEscherichia coli

The FIS protein (factor for inversion stimulation) is known to stimulate site-specific recombination processes, such as the inversion of the G segment of bacteriophage Mu, by binding to specific enhancer sequences. It has also been shown to activate transcription from rRNA promoters both in vitro and in vivo. We have identified a specific binding site for FIS in the center of the origin of chromosomal DNA replication, oriC. The DNA bends upon FIS binding. Occupation of the FIS site and binding of DnaA, the initiator protein, to its adjacent binding site (R3) are mutually exclusive. A fis mutant strain can not be efficiently transformed with plasmids which carry and replicate from oriC, suggesting that FIS is required for minichromosome replication.

The complex of oriC DNA with the DnaA initiator protein

We describe several experimental approaches relating to the early steps in the initiation of DNA replication at oriC. 1) A matrix is given which enables calculation of the relative affinity of DnaA boxes for DnaA protein; 2) base changes within single Dna A boxesiin oriC have little effect on oriC function; 3) mutations which change the distance between DnaA boxes inactivate oriC, but changes by one helical turn (+ and - ) result in near wild-type oriC activity; 4) a Fis binding site was located at oriC coordinates 206–220; 5) KMnO 4 probing demonstrates Dna-A-dependent unwinding in the left part of oriC in vivo and in vitro.

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.

Potential binding sites of the trans-activator FIS are present upstream of all rRNA operons and of many but not all tRNA operons

FIS, the Escherichia coli protein that stimulates the inversion of various DNA segments by binding to a recombinational enhancer, trans-activates a number of stable RNA operons and binds to the upstream activator sequence (UAS) of these operons (Nilsson et al. (1990) EMBO J. 9, 727). In a search for potential FIS-binding sites we have compared UASs of other stable RNA operons with a consensus FIS-binding sequence, compiled by comparing recombinational enhancers. Such sites can thus be recognized upstream of all rRNA and 13 tRNA operons. Matching with the consensus sequence varied, suggesting that the affinity of FIS for the sites differed. Accordingly, FIS binding to an upstream sequence of the metY(nusA) operon was found to be weaker than that to the UAS of the thrU(tufB) operon. No FIS binding sites were found upstream three tRNA operons.