Integration Host Factor Binding Research Articles

The Binding Parameters of Integration Host Factor to the Lambda Phage Cosi1 Site and Holliday Junction DNA

Integration Host Factor (IHF) is a heterodimeric DNA-binding protein produced by E.coli. IHF interacts with the minor groove and phosphodiester backbone of the DNA to create sharp bends in the DNA and is often referred to as an architectural protein. Although the protein binds sequence-specifically, it also recognizes DNA conformation. Prokaryotic cellular processes involving IHF include viral integration, replication and activation of gene expression. We have recently shown that IHF exhibits high binding affinity for the Holliday or four-way junction DNA (4WJ), an intermediate in homologous recombination where strands from different chromosomes cross over. Förster Resonance Energy Transfer (FRET), where the 5'-ends of DNA strands are labeled with fluorophores, is used to determine the conformational changes of the 4WJ when interacting with DNA-binding proteins. These measurements have shown that the 4WJ opens upon binding with IHF, which is consistent with data promoting the protein's involvement in recombinase action. Subsequent FRET experiments performed confirm this observation, and provide a value for the efficiency of binding and the degree of distortion induced. Interactions between IHF and the cos I1 site of DNA from the lambda bacteriophage will also be explored. It is known that IHF induces bending at cos sites. The affinity of binding to Δ cos DNA is compared to that between IHF and its consensus sequence (H1) in E. coli DNA. Gel mobility shift assay (GMSA) and solutions experiments will measure the binding and affinity of IHF with the cos I1 site. FRET experiments with labeled cosI1 DNA will reveal the distortion induced upon IHF binding and these results will be compared to those with IHF binding to the H1 site.

Physical Organization of DNA by Multiple Non-Specific DNA-Binding Modes of Integration Host Factor (IHF)

The integration host factor (IHF) is an abundant nucleoid-associated protein and an essential co-factor for phage λ site-specific recombination and gene regulation in E. coli. Introduction of a sharp DNA kink at specific cognate sites is critical for these functions. Interestingly, the intracellular concentration of IHF is much higher than the concentration needed for site-specific interactions, suggesting that non-specific binding of IHF to DNA plays a role in the physical organization of bacterial chromatin. However, it is unclear how non-specific DNA association contributes to DNA organization. By using a combination of single DNA manipulation and atomic force microscopy imaging methods, we show here that distinct modes of non-specific DNA binding of IHF result in complex global DNA conformations. Changes in KCl and IHF concentrations, as well as tension applied to DNA, dramatically influence the degree of DNA-bending. In addition, IHF can crosslink DNA into a highly compact DNA meshwork that is observed in the presence of magnesium at low concentration of monovalent ions and high IHF-DNA stoichiometries. Our findings provide important insights into how IHF contributes to bacterial chromatin organization, gene regulation, and biofilm formation.

Multiple Binding Modes and DNA Organization by Non-Specific Binding of the E. Coli Nucleoid Associated Protein IHF

The integration host factor (IHF) protein is an abundant E.Coli nucleoid protein. It is most well known as an essential cofactor in the process of phage Δ site-specific recombination where it binds to highly specific DNA sequences with a dissociation coefficient of less than 0.1 nM. Surprisingly, the intra-cellular concentration of IHF is in the range of 10 - 100 uM, extraordinarily larger than the need by its site-specific function. This raises an important question regarding the roles of non-specific binding of IHF that are yet to be understood. Here, we report several novel findings that IHF can bind to DNA with multiple distinct modes under physiological buffer conditions, and can organize DNA into highly condensed complex structures. Our findings have broad implications and highlight a role of IHF as an essential chromosomal DNA packaging protein.

Involvement of the Global Crp Regulator in Cyclic AMP-Dependent Utilization of Aromatic Amino Acids by Pseudomonas putida

The phhAB operon encodes a phenylalanine hydroxylase involved in the conversion of L-phenylalanine into L-tyrosine in Pseudomonas putida. The phhAB promoter is transcribed by RNA polymerase sigma-70 and is unusual in that the specific regulator PhhR acts as an enhancer protein that binds to two distant upstream sites (-75 to -92 and -132 to -149). There is an integration host factor (IHF) binding site that overlaps the proximal PhhR box, and, consequently, IHF acts as an inhibitor of transcription. Use of L-phenylalanine is compromised in a crp-deficient background due to reduced expression from the phhAB promoter. Electrophoretic mobility shift assays and DNase I footprinting assays reveal that Crp binds at a site centered at -109 only in the presence of cyclic AMP (cAMP). We show, using circular permutation analysis, that the simultaneous binding of Crp/cAMP and PhhR bends DNA to bring positive regulators and RNA polymerase into close proximity. This nucleoprotein complex promotes transcription from phhA only in response to L-phenylalanine.

Functional analysis of the integration host factor site of the σ54Pu promoter of Pseudomonas putida by in vivo UV imprinting

The integration host factor (IHF) of Pseudomonas putida connects cell growth to transcriptional activity of distinct promoters. The IHF site of the σ(54) promoter Pu of the TOL (m-xylene biodegradation) plasmid pWW0 of P. putida has been examined to define experimentally a relationship between occupation of the promoter by this factor, the biological activity of the protein and the tolerance of the target site to single-base changes through the bound DNA core sequence. The use of an in vivo high-intensity UV imprinting procedure to examine such an occupation of Pu by IHF allowed inspection of the interplay between the factor and cognate site variants under the physiologically relevant conditions of monocopy gene dosage. The resulting data were merged in a structural model for establishing key features of the IHF-DNA interaction. A functional consensus for first-order IHF binding was instrumental for a genome-wide survey of sequences with potential regulatory value. This search revealed that very few, if any, of the maximum 330 sites within intergenic regions were placed in locations controlling expression of central metabolic genes. It thus seems that the IHF regulon of P. putida has a degree of functional specialization that is not evenly distributed through all gene categories.

Variation of the intercalating proline in artificial peptides mimicking the DNA binding and bending IHF protein.

The integration host factor (IHF) is a protein which sequence specifically induces a bend of double-stranded DNA by more than 160°. Based on IHF as lead structure, a peptide mimic was introduced resembling the positively charged body of the protein by a lysine dendrimer and the minor groove recognition loop by a cyclopeptide. The proline located close to the tip of the recognition loop intercalates between the base pair plane. It was modified in order to evaluate the influence of the side chain residue with respect to size (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), aromaticity (phenylalanine), conformation of the five-membered ring [(4R)-fluoroproline, (4S)-fluoroproline, 3,4-dehydroproline], and the peptide backbone conformation (α-methylproline) on binding dsDNA and bending the double strand. Binding and bending studies were carried out by fluorescence resonance energy transfer experiments and gel electrophoresis using DNA sequences prepared by PCR with the IHF binding site in central or terminal position. Whereas aromatic residues and α-methylproline were not tolerated as proline substitute, incorporation of (4S)-fluoroproline and 3,4-dehydroproline provided enhanced binding.

Nonspecific DNA Binding and Bending by HU αβ: Interfaces of the Three Binding Modes Characterized by Salt-Dependent Thermodynamics

Previous isothermal titration calorimetry (ITC) and Förster resonance energy transfer studies demonstrated that Escherichia coli HU αβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34-bp mode that interacts with DNA in large (> 34 bp) gaps between bound proteins, reversibly bending it by 140 o and thereby increasing its flexibility, and two weaker, modestly cooperative small site-size modes (10 bp and 6 bp) that are useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and we deduce that DNA in the 34-bp mode is bent around—but not wrapped on—the body of HU, in contrast to specific binding of integration host factor. Analyses of binding isotherms (8-bp, 15-bp, and 34-bp DNA) and initial binding heats (34-bp, 38-bp, and 160-bp DNA) reveal that all three modes have similar log–log salt concentration derivatives of the binding constants (Sk i ) even though their binding site sizes differ greatly; the most probable values of Sk i on 34-bp DNA or larger DNA are − 7.5 ± 0.5. From the similarity of Sk i values, we conclude that the binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent DNA 34-bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6-bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Sk i values are proposed.

Repression of aerobic leukotoxin transcription by integration host factor in Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans has been implicated as the primary etiologic agent in localized aggressive periodontitis. This bacterium produces a leukotoxin which may help the bacterium evade the host immune response. Leukotoxin transcription is induced when A. actinomycetemcomitans is grown anaerobically, as in the periodontal pocket. Previously, a 35 bp oxygen-response-element (ORE) was shown to be responsible for oxygen regulation at the leukotoxin promoter. However, the gene’s transcription is not controlled by Fnr or ArcA, the major oxygen regulators in other bacteria. To identify the potentially novel protein(s) that regulate leukotoxin transcription, protein extracts of A. actinomycetemcomitans were tested for ORE binding by mobility shift assays; one ORE-specific binding complex was found. Standard fractionation protocols and protein sequencing identified the ORE binding protein as integration host factor (IHF). DNaseI protection assays showed that the IHF binding site overlaps the first half of the ORE. To assess the effect of IHF on leukotoxin synthesis, an A. actinomycetemcomitans deletion mutant in ihfB was constructed and characterized. Interestingly, leukotoxin RNA and protein synthesis was de-repressed in the ihf mutant, although leukotoxin synthesis in still oxygen-regulated in the mutant cells. Thus, IHF plays a direct role in repressing leukotoxin transcription, but another protein is also involved in regulating leukotoxin expression in response to oxygen.

Direct Interaction between Quorum-sensing Regulator SmcR and RNA Polymerase Is Mediated by Integration Host Factor to Activate vvpE Encoding Elastase in Vibrio vulnificus

It has been suggested that quorum sensing is an important signal transduction system regulating the expression of numerous virulence genes in bacterial pathogens. We previously revealed that SmcR, a LuxR homologue of Vibrio vulnificus, activates promoter S, an RpoS-dependent promoter of vvpE encoding a potential virulence factor elastase and binds in vitro to a binding site centered at -196.5. In this study, chromatin immunoprecipitation assays and promoter deletion analyses demonstrated that SmcR binds to the vvpE regulatory region in vivo and directly interacts with RNAP for activation of the vvpE expression. A search for regulatory genes involved in the regulation of elastase production singled out ihfA, which encodes for a subunit of integration host factor (IHF). Levels of both elastase activity and vvpE transcript decreased significantly as a result of inactivation of ihfA, and primer extension analyses demonstrated that IHF regulates the vvpE transcription by activating PS. Direct binding of IHF to the two distinct binding sites centered at -174 and -131, respectively, was determined using an electrophoretic mobility shift assay and a DNase I protection assay. Chromatin immunoprecipitation assays revealed that the interaction of SmcR with RNAP in vivo was mediated by IHF. Collectively, the results proposed a model whereby IHF positions SmcR to contact RNAP by looping the vvpE regulatory DNA, thus allowing precise control of the expression level of VvpE during the pathogenesis of V. vulnificus.

Platform for in situ real-time measurement of protein-induced conformational changes of DNA

A platform for in situ and real-time measurement of protein-induced conformational changes in dsDNA is presented. We combine electrical orientation of surface-bound dsDNA probes with an optical technique to measure the kinetics of DNA conformational changes. The sequence-specific Escherichia coli integration host factor is utilized to demonstrate protein-induced bending upon binding of integration host factor to dsDNA probes. The effects of probe surface density on binding/bending kinetics are investigated. The platform can accommodate individual spots of microarrayed dsDNA on individually controlled, lithographically designed electrodes, making it amenable for use as a high throughput assay.

Binding and Bending Parameters of Integration Host Factor to Four-Way Holliday Junction

Binding and Bending Parameters of Integration Host Factor to Four-Way Holliday JunctionIntegration host factor (IHF) is a small heterodimeric protein that sequence specifically binds the minor groove of DNA and facilitates a bend of nearly 180 degrees. This bending is crucial for cellular processes such as recombination, replication and transcription. Previous work in the Mukerji lab characterized the binding properties of the structurally similar but sequence non-specific DNA-binding protein HU, to duplex and Holliday junction DNA. This research demonstrated that HU binds to the central region of the junction with nanomolar affinity and prefers the stacked form of the Holliday junction. Given the similarities in structure and function of HU and IHF, we elected to study the binding capability of IHF to the Holliday junction. We have compared the binding affinity of IHF for Holliday junction DNA with its binding affinity to duplex DNA with and without a consensus sequence. Binding measurements were performed using florescence intensity and anisotropy methods and confirmed with the gel mobility shift assay. All binding assays established that IHF binds to the Holliday junction lacking a consensus sequence with high affinity (∼3 nanomolar Kd) similar to HU, suggesting that both proteins might recognize and bind similar structural aspects of the Holliday junction. Moreover, anisotropy affinity measurements demonstrated that IHF binds the Holliday junction with similar affinity as it does to duplex DNA containing its consensus sequence. The span of binding capabilities exhibited by IHF indicate that it can function both as a specific and non-sequence specific DNA binding protein.

A Novel Mechanism of Transposon-Mediated Gene Activation

Transposable Insertion Sequences (IS elements) have been shown to provide various benefits to their hosts via gene activation or inactivation under stress conditions by appropriately inserting into specific chromosomal sites. Activation is usually due to derepression or introduction of a complete or partial promoter located within the element. Here we define a novel mechanism of gene activation by the transposon IS5 in Escherichia coli. The glycerol utilization operon, glpFK, that is silent in the absence of the cAMP-Crp complex, is activated by IS5 when inserted upstream of its promoter. High-level expression is nearly constitutive, only mildly dependent on glycerol, glucose, GlpR, and Crp, and allows growth at a rate similar to or more rapid than that of wild-type cells. Expression is from the glpFK promoter and dependent on (1) the DNA phase, (2) integration host factor (IHF), and (3) a short region at the 3′ end of IS5 harboring a permanent bend and an IHF binding site. The lacZYA operon is also subject to such activation in the absence of Crp. Thus, we have defined a novel mechanism of gene activation involving transposon insertion that may be generally applicable to many organisms.

IHF-binding sites inhibit DNA loop formation and transcription initiation

Transcriptional activation of enhancer and σ54-dependent promoters requires efficient interactions between enhancer-binding proteins (EBP) and promoter bound σ54-RNA polymerase (Eσ54) achieved by DNA looping, which is usually facilitated by the integration host factor (IHF). Since the lengths of the intervening region supporting DNA-loop formation are similar among IHF-dependent and IHF-independent promoters, the precise reason(s) why IHF is selectively important for the frequency of transcription initiation remain unclear. Here, using kinetic cyclization and in vitro transcription assays we show that, in the absence of IHF protein, the DNA fragments containing an IHF-binding site have much less looping-formation ability than those that lack an IHF-binding site. Furthermore, when an IHF consensus-binding site was introduced into the intervening region between promoter and enhancer of the target DNA fragments, loop formation and DNA-loop-dependent transcriptional activation are significantly reduced in a position-independent manner. DNA-looping-independent transcriptional activation was unaffected. The binding of IHF to its consensus site in the target promoters clearly restored efficient DNA looping formation and looping-dependent transcriptional activation. Our data provide evidence that one function for the IHF protein is to release a communication block set by intrinsic properties of the IHF DNA-binding site.

Initiator titration complex formed at datA with the aid of IHF regulates replication timing in Escherichia coli

The initiation of replication in Escherichia coli is negatively controlled by a mechanism referred to as 'initiator titration', a process by which the initiator protein, DnaA, is titrated to newly replicated binding sequences on the chromosome to reduce the initiation potential for replication. Initiator titration occurs predominantly at the datA locus that binds exceptionally large amounts of DnaA molecules to prevent aberrant initiations. We found that this was enabled by integration host factor (IHF). Within datA, there is a consensus IHF recognition sequence between the two DnaA recognition sequences (DnaA boxes) essential for its function. Binding of IHF to this site was demonstrated both in vitro and in vivo. Disruption of the core sequence in the consensus of the IHF-binding resulted in increased origin concentration as observed in Delta datA cells. Furthermore, the number of DnaA molecules bound to datA was reduced in cells carrying a disruption in the IHF-binding core sequence. The IHF-binding site and the essential DnaA boxes had to be located at a proper distance and orientation to maintain the accurate initiation timing. Therefore, IHF is a unique element in the control of replication initiation that acts negatively at datA, while known to act as a positive regulator at oriC.

Ion Exchange in the Nonspecific Bimolecular Association and the Unimolecular DNA Bending in Specific Binding of IHF to DNA

Protein-DNA interactions are strongly modulated by salt. Previous studies on the equilibrium binding of a cognate DNA sequence H′ to integration host factor (IHF), an architectural protein from E. coli that bends its cognate site by nearly 180°, have shown that the slope SKD of ∼8 on a log(KD) versus log([KCl]) plot depends on the anion type, suggesting that both release of counterions from the DNA and the uptake and release of ions from the protein must be playing a role. Here, we probe the effect of [KCl] on the bimolecular association/dissociation as well as the unimolecular bending/unbending rates, by monitoring the relaxation kinetics of the complex between IHF and ∼35-bp long H' substrate end-labeled with a FRET pair, in response to a laser temperature-jump. Our results and analysis reveal two notable results. First, that the unimolecular bending step is nearly independent of [KCl]. Second, that the bulk of the salt-dependence appears in the nonspecific association/dissociation step, with the equilibrium constant for that step accounting for more than half of the total SKD observed. The latter result is in contrast to what one might expect if counterion release from the DNA was the dominant contribution to the salt-dependence, since the extent to which the H' substrate makes contact with the protein in the fully wrapped specific complex is significantly greater than in the nonspecific complex. One possible scenario is that counterion release is the dominant term in the formation of the nonspecific complex, whereas in the transition from the nonspecific to the specific complex, the extent of the counterion release is masked by the uptake and release of ions by the protein, as a result of conformational rearrangements in the protein.

Integration Host Factor (IHF) Dictates the Structure of Polyamine-DNA Condensates: Implications for the Role of IHF in the Compaction of Bacterial Chromatin

Integration host factor (IHF), a nucleoid-associated protein in bacterial cells, is implicated in a number of chromosomal functions including DNA compaction. IHF binds to all duplex DNA with micromolar affinity and at sequence-specific sites with much higher affinity. IHF is known to induce sharp bends in the helical axis of DNA in both modes of binding, but the role of IHF in controlling DNA condensation within bacterial cells has remained undetermined. Here we demonstrate that IHF influences the morphology of DNA condensed by polyamines in vitro. In the absence of IHF, spermidine and spermine condense DNA primarily into toroidal structures, whereas in the presence of IHF, polyamines condense DNA primarily into rodlike structures. Computer simulations of DNA condensation in the absence and presence of IHF binding lend support to our model in which DNA bending proteins, such as IHF and HU, promote the condensation of DNA into rodlike structures by providing the free energy necessary to bend DNA at the ends of linear bundles of condensed DNA. We propose that a common function of IHF and HU in bacterial cells is to facilitate DNA organization in the nucleoid by the introduction of sharp bends in chromosomal DNA.

Regulation by Nucleoid-Associated Proteins at the Escherichia coli nir Operon Promoter

The Escherichia coli K-12 nir operon promoter can be fully activated by binding of the regulator of fumarate and nitrate reduction (FNR) to a site centered at position -41.5 upstream of the transcript start, and this activation is modulated by upstream binding of the integration host factor (IHF) and Fis (factor for inversion stimulation) proteins. Thus, transcription initiation is repressed by the binding of IHF and Fis to sites centered at position -88 (IHF I) and position -142 (Fis I) and activated by IHF binding to a site at position -115 (IHF II). Here, we have exploited mutational analysis and biochemistry to investigate the actions of IHF and Fis at these sites. We show that the effects of IHF and Fis are position dependent and that IHF II functions independently of IHF I and Fis I. Using in vitro assays, we report that IHF and Fis repress transcription initiation by interfering with RNA polymerase binding. Differences in the upstream IHF and Fis binding sites at the nir promoter in related enteric bacteria fix the level of nir operon expression under anaerobic growth conditions.

Integration Host Factor Positively Regulates Virulence Gene Expression in Vibrio cholerae

Virulence gene expression in Vibrio cholerae is dependent upon a complex transcriptional cascade that is influenced by both specific and global regulators in response to environmental stimuli. Here, we report that the global regulator integration host factor (IHF) positively affects virulence gene expression in V. cholerae. Inactivation of ihfA and ihfB, the genes encoding the IHF subunits, decreased the expression levels of the two main virulence factors tcpA and ctx and prevented toxin-coregulated pilus and cholera toxin production. IHF was found to directly bind to and bend the tcpA promoter region at an IHF consensus site centered at position -162 by using gel mobility shift assays and DNase I footprinting experiments. Deletion or mutation of the tcpA IHF consensus site resulted in the loss of IHF binding and additionally disrupted the binding of the repressor H-NS. DNase I footprinting revealed that H-NS protection overlaps with both the IHF and the ToxT binding sites at the tcpA promoter. In addition, disruption of ihfA in an hns or toxT mutant background had no effect on tcpA expression. These results suggest that IHF may function at the tcpA promoter to alleviate H-NS repression.

Small mobile sequences in bacteria display diverse structure/function motifs

Small repeat sequences in bacterial genomes, which represent non-autonomous mobile elements, have close similarities to archaeon and eukaryotic miniature inverted repeat transposable elements. These repeat elements are found in both intergenic and intragenic chromosomal regions, and contain an array of diverse motifs. These can include DNA sequences containing an integration host factor binding site and a proposed DNA methyltransferase recognition site, transcribed RNA secondary structural motifs, which are involved in mRNA regulation, and translated open reading frames found fused to other open reading frames. Some bacterial mobile element fusions are in evolutionarily conserved protein and RNA genes. Others might represent or lead to creation of new protein genes. Here we review the remarkable properties of these small bacterial mobile elements in the context of possible beneficial roles resulting from random insertions into the genome.

Molecular Beacon-Equilibrium Cyclization Detection of DNA-Protein Complexes

Molecular beacon detection of equilibrium cyclization (MBEC) is a novel, high sensitivity technique that can allow DNA-protein complex formation to be studied under diverse conditions in a cost effective and rapid manner that can be adapted to high throughput screening. To demonstrate the ease and utility of applying MBEC to the investigation of the K D values of protein-DNA complexes, the sequence-specific Escherichia coli integration host factor (IHF) protein has been used as a test system. Competition between a labeled MBEC DNA construct and unlabeled duplex DNA for IHF binding allows the determination of K D values as a function of the DNA duplex sequence. This allows sequence specificity to be monitored while using only a single molecular beacon-labeled DNA. The robustness of MBEC for monitoring protein-DNA complex formation has been further demonstrated by determining the K D values as a function of salt concentration to investigate the net number of salt bridges formed in sequence-specific and -nonspecific IHF-DNA complexes. These MBEC results have been compared with those from other approaches.