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Results for “Integration Host Factors”

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At least 271 records · Page 15Linked to original sources

PubMed2003

Deciphering environmental signal integration in sigma54-dependent promoters with a simple mathematical model.

A mathematical model was developed to describe the physiological co-regulation of two Pseudomonas sigma54-dependent promoter/regulator systems, Pu/XylR and Po/DmpR of Pseudomonas strains mt2 and CF600, respectively. Five ordinary differential equations and six algebraic equations were developed to describe the following processes of transcription initiation: binding of the activator protein to the upstream activating sequence, union of the sigma factor with the core polymerase, formation of the open complex, and escape of the transcription machinery from the promoter region. In addition, growth-phase control of the integration host factor (IHF), sigma-70 regulation during stationary phase, and the contribution of (p)ppGpp to both sigma factor selectivity and promoter escape were hypothesized. By including any three of these four effects, the model predicted that expression from both promoters is repressed during exponential growth and sharply increases as the cells enter stationary phase. The difference in behavior of the two systems during overexpression of either sigma54 or (p)ppGpp could be explained by different values of two model parameters. To accurately represent the behavior of both promoters in (p)ppGpp null strains, an additional parameter must be varied. Although numerical data available for this system is scarce, the model has proved useful for helping to interpret the experimental observations and to evaluate four hypotheses that have been proposed to explain the phenomenon of exponential silencing.

Bacterial Proteins↗

PubMed1991

A switch in the formation of alternative DNA loops modulates lambda site-specific recombination.

The virally encoded Xis protein is one of the components in the site-specific recombination reactions of bacteriophage lambda. It is required for excisive recombination and inhibits integrative recombination. The mechanism of Xis inhibition of the integration reaction was investigated by methylation protection assays (footprinting analyses) in conjunction with recombination assays. Xis is shown to mediate the formation of a specific attP looped structure involving cooperative and competitive long-range interactions among integrase, integration host factor, and Xis proteins. This higher-order structure precludes supercoiled attP from engaging in the productive partner interactions that lead to execution of the first strand exchange in integrative recombination. In addition to its previously characterized role in excision, Xis-induced DNA bending is postulated to act as a regulatory switch (in an alternative loop mechanism) that converts the attP intasome from an integrative-competent complex to a nonreactive one.

Bacterial Proteins↗

PubMed1998

The role of supercoiling in mycobacteriophage L5 integrative recombination.

The genome of temperate mycobacteriophage L5 integrates into the chromosomes of its hosts, including Mycobacterium smegmatis , Mycobacterium tuberculosis and bacille Calmette-Guérin. This integrase-mediated site-specific recombination reaction occurs between the phage attP site and the mycobacterial attB site and requires the mycobacterial integration host factor. Here we examine the role of supercoiling in this reaction and show that integration is stimulated by DNA supercoiling but that supercoiling of either the attP or the attB substrate enhances recombination. Supercoiling thus facilitates a post-synaptic recombination event. We also show that, while supercoiling is not required for the production of a recombinagenic intasome, a mutant attP DNA deficient in binding of the host factor acquires a dependence on supercoiling for intasome formation and recombination.

Bacterial Proteins↗

PubMed1998

GATC motifs may alter the conformation of DNA depending on sequence context and N6-adenine methylation status: possible implications for DNA-protein recognition.

As part of our analysis of the role of a uniquely clustered set of dam methylation sites (the motif GATC) within the origin of DNA replication in Escherichia coli, we have studied the effect of GATCs in various methylation states on the intrinsic curvature of DNA. We have designed a set of DNA linkers and used commercially available linkers containing GATC motifs. The linkers were ligated and the electrophoretic mobility of the resulting multimers in different states of methylation was tested relative to reference fragments. We report that properly phased GATCs in certain sequence environments modulate DNA curvature and that these effects may be enhanced by N6-adenine methylation of the GATCs. These structural alterations may in turn affect DNA-protein interactions, especially those involving proteins that rely on both primary sequence and structure for recognition. We present an example, where introduction of a GATC within an integration host factor (IHF) binding site, which does not alter the consensus sequence, reduces the binding affinity of the protein for the modified site.

Adenine↗

PubMed1987

Tn10 transposition and circle formation in vitro.

We describe a cell-free system that promotes Tn10 transposition and transposon circle formation, a related intramolecular event. Tn10 circle formation in vitro has been characterized in detail, and is shown to require a supercoiled substrate and to proceed in the absence of ATP. The reaction requires Tn10 transposase protein, and either of two E. coli proteins, integration host factor (IHF) and HU, which are small DNA binding proteins that change the conformation of DNA. Tn10 is composed of inverted repeats of insertion sequence IS10. Pair-wise combinations of the IS10 "outside" and "inside" ends mediate distinct classes of rearrangements in vivo, and they exhibit different reaction requirements in vitro. In contrast to the Tn10 reaction, which involves two outside ends, circle formation with two inside ends proceeds with a transposase fraction alone, in the absence of added host factors, and is inhibited by methylation of the dam site within each terminus.

Adenosine Triphosphatases↗

PubMed1996

The flavohaemoglobin (HMP) of Escherichia coli generates superoxide in vitro and causes oxidative stress in vivo.

Purified flavohaemoglobin (HMP) of Escherichia coli reduces Fe(III) in a superoxide dismutase (SOD)-sensitive reaction, demonstrating superoxide anion generation during aerobic NADH oxidation. In vivo, sodA-lacZ fusion activity was increased 3-fold by introducing plasmid pPL341, containing the hmp gene, or by growth with paraquat. The effects were additive and SOXS-dependent. Thus HMP activity causes oxidative stress in vivo. Activities of sodA-lacZ and hmp-lacZ fusions were stimulated in a himA mutant, demonstrating repression of both promoters by integration host factor (IHF), but the effects of pPL341 on sodA-lacZ activity were not due to titration of IHF by the hmp promoter.

Aerobiosis↗

PubMed2002

Site-specific recombination in eukaryotic cells mediated by mutant lambda integrases: implications for synaptic complex formation and the reactivity of episomal DNA segments.

Mutant lambda integrases catalyze site-specific DNA recombination in the absence of accessory factors IHF, XIS, and negative DNA supercoiling. Here we investigate the effects that a human cellular environment exerts on these reactions in order to (i) gain further insights into mechanistic aspects of recombination in eukaryotic cells and (ii) to further develop the Int system for biotechnological applications. First, we compared intra- and intermolecular integrative as well as excisive recombination pathways on episomal substrates after co-transfection with recombinase expression vectors. Our results demonstrate that, within 24 hours after transfection, intermolecular recombination by mutant integrase is at least as efficient as intramolecular recombination. Second, a significant intermolecular recombination activity was observed between two copies of a recombination site containing only the 21 bp comprising core-type DNA sequence. This basic activity was stimulated several-fold when arm-type DNA sequences were present in addition to core sites. Therefore, one recombination pathway in human cells involves mutant integrases bound solely at core sites, which is reminiscent of the Flp/FRT and Cre/loxP pathways. The stimulatory effect of arm-type sequences could be explained by an increase in integrase concentration in the vicinity of core sites. We show, in addition, that an N-terminal truncated mutant integrase exhibited only a very weak recombinogenic activity in a eukaryotic background. This result strengthens a functional role for the N-terminal domain in recombination in addition to its arm-type DNA-binding activity. Finally, we demonstrate that low level integrative recombination by wild-type integrase is stimulated when purified integration host factor is co-transfected. This corroborates our previous conclusion that sufficient amounts of eukaryotic protein co-factors, which could functionally replace IHF, are not present in human cells. It also provides a potential means to control site-specific recombination in eukaryotic cells.

Attachment Sites, Microbiological↗

PubMed2000

sigma factor selectivity of Escherichia coli RNA polymerase: role for CRP, IHF and lrp transcription factors.

osmY is a stationary phase-induced and osmotically regulated gene in Escherichia coli that requires the stationary phase RNA polymerase (Esigma(S)) for in vivo expression. We show here that the major RNA polymerase, Esigma(70), also transcribes osmY in vitro and, depending on genetic background, even in vivo. The cAMP receptor protein (CRP) bound to cAMP, the leucine-responsive regulatory protein (Lrp) and the integration host factor (IHF) inhibit transcription initiation at the osmY promoter. The binding site for CRP is centred at -12.5 from the transcription start site, whereas Lrp covers the whole promoter region. The site for IHF maps in the -90 region. By mobility shift assay, permanganate reactivity and in vitro transcription experiments, we show that repression is much stronger with Esigma(70) than with Esigma(S) holoenzyme. We conclude that CRP, Lrp and IHF inhibit open complex formation more efficiently with Esigma(70) than with Esigma(S). This different ability of the two holoenzymes to interact productively with promoters once assembled in complex nucleoprotein structures may be a crucial factor in generating sigma(S) selectivity in vivo.

Bacterial Proteins↗

PubMed1992

In vitro interactions of the histone-like protein IHF with the algD promoter, a critical site for control of mucoidy in Pseudomonas aeruginosa.

The ability of the histone-like element Integration Host Factor (IHF) to interact with the algD promoter was investigated. IHF from Escherichia coli was found to bind to the algD promoter and to form multiple protein-DNA complexes in gel mobility shift DNA binding assay. The highest affinity binding site for IHF was mapped by DNaseI footprinting analysis. This site spanned nucleotides -50 to -85 relative to the algD mRNA start site and overlapped a sequence matching the IHF consensus sequence WATCAANNNNTTR in 12 out of 13 base pairs. Previous studies have shown that deletion of sequences including a portion of this site adversely affects algD promoter activity. IHF binding to the algD promoter induced DNA bending. Western blot analysis with antibodies against E. coli IHF detected a cross-reactive protein of a similar molecular mass in Pseudomonas aeruginosa, suggesting the presence of an analogous factor in this organism.

Amino Acid Sequence↗

PubMed1995

Characterization of the binding of HU and IHF, homologous histone-like proteins of Escherichia coli, to curved and uncurved DNA.

The binding of E. coli histone-like protein HU to curved and uncurved DNA fragments containing adenine tracts was characterized by relative binding affinity assay, and compared with that of other homologous histone-like protein integration host factor (IHF). Both HU and IHF have about 3- to 5-fold higher affinity for overall curved DNA fragments such as (A6N4)11 and (A3T3N4)12 compared to a standard duplex fragment with mixed sequence. The binding manner of HU to the curved fragments was highly cooperative. However, loss of overall curvature for shorter fragments (< approximately 100 bp) reduced the preference of HU binding to curved (A3T3N4)n over uncurved (T3A3N4)n, indicating that the binding specificity of HU to curved DNA is length-dependent. Thus, the curved DNA configuration of the whole molecule facilitates the binding of several HU molecules to form the hierarchy of HU-DNA complex. Furthermore, it was shown that HU and IHF bind less well to (A6N9)n, which has a zig-zag straight structure, whereas they preferentially bind to uncurved (T3A3N4)14. These results suggested that not only intrinsically overall curvature but also the preferred orientations for DNA bending in the protein-DNA complex are important factors for affinities of HU and IHF.

Bacterial Proteins↗

PubMed1993

Two-dimensional gel selection of protein binding sites on DNA.

We have developed a gel electrophoretic approach for visualizing and cloning protein binding sites from complete genomes. This system consists of a simple two-dimensional band shift, in which protein-DNA complexes are retarded in the first dimension, performed at low temperature, and disrupted in the second dimension, performed at high temperature. We present here results obtained with the integration host factor (IHF) and cAMP receptor protein (CRP) proteins of Escherichia coli, and discuss some of the important methodological aspects of the technique.

Amino Acid Sequence↗

PubMed1997

Long-range effects in a supercoiled DNA domain generated by transcription in vitro.

The translocation of a transcription complex can transiently introduce positive and negative superhelical windings into the template DNA. To gain further insight into this dynamic DNA supercoiling mechanism and its possible involvement in biological processes, we employed an in vitro system in which site-specific recombination by gammadelta resolvase is topologically coupled to transcription-induced negative supercoiling. Our kinetic experiments suggest that recombination is closely linked to the process of supercoiling by transcription. We utilized the known high speed at which two resolvase-bound recombination sites can pair to form a synaptic complex in kinetic experiments with DNA substrates containing three recombination sites. Our data provide evidence for the existence of a transient gradient of negative supercoiling. Such a gradient seems to be predominantly a consequence of DNA double helix rotation behind a translocating RNA polymerase and originates within a broad region up to two kilobase-pairs upstream of the transcriptional start site. We further demonstrate that the topological coupling between transcription and recombination is not affected when the DNA-bending protein integration host factor from E. coli is bound to multiple sites within the phage lambda attachment region. We discuss implications of our in vitro findings with respect to possible in vivo functions of the dynamic nature of transcription-induced supercoiling.

Bacterial Proteins↗

PubMed1995

Enhanced activity of the bacteriophage lambda PL promoter at low temperature.

We found that the activity of the phage lambda PL promoter is inversely dependent on temperature. Both in vivo and in vitro transcription assays revealed that the rather unique temperature response of PL is a sum of an intrinsic property of the promoter and its activation by integration host factor. We also found that at low temperature, phage lambda can lysogenize efficiently but cannot complete the lytic cycle. We hypothesize that by sensing the low environmental temperature the PL promoter plays a role in determining the direction of phage lambda development.

Bacterial Proteins↗

PubMed1995

Temperature tolerance of hydrogenase expression in Alcaligenes eutrophus is conferred by a single amino acid exchange in the transcriptional activator HoxA.

Expression of the soluble (SH) and membrane-bound (MBH) hydrogenases in the facultatively lithoautotrophic bacterium Alcaligenes eutrophus is dependent on the transcriptional activator HoxA and the alternative sigma factor sigma 54. Deletion analysis revealed that a region 170 bp upstream of the transcriptional start of the SH operon is necessary for high-level promoter activity. Mobility shift assays with DNA fragments containing the SH upstream region and purified beta-galactosidase-HoxA fusion protein isolated from Escherichia coli or authentic HoxA isolated by immunoaffinity chromatography from A. eutrophus failed to detect specific binding. In contrast, A. eutrophus extracts enriched for HoxA by heparin-Sepharose chromatography and ammonium sulfate fractionation produced a weak but discrete shift in the mobility of the target DNA. This effect was not observed with comparable extracts prepared from hoxA mutants. A similar experiment using antibodies against HoxA confirmed that HoxA was responsible for the observed mobility shift. Extracts prepared from a temperature-tolerant mutant of A. eutrophus gave a stronger retardation than did those from the wild type. Unlike the wild type, the hox(Tr) mutant is able to grow with hydrogen at temperatures above 33 degrees C because of a mutation in the regulatory gene hoxA. In this paper, we show that a single amino acid substitution (Gly-468-->Val) in the C-terminal part of HoxA is responsible for temperature tolerance. The SH upstream region also contains sequence motifs resembling the E. coli integration host factor (IHF) binding site, and purified E. coli IHF protein shifted the corresponding indicator fragment.

Alcaligenes↗

PubMed1998

A protein-induced DNA bend increases the specificity of a prokaryotic enhancer-binding protein.

Control of transcription in prokaryotes often involves direct contact of regulatory proteins with RNA polymerase from binding sites located adjacent to the target promoter. Alternatively, in the case of genes transcribed by Escherichia coli RNA polymerase holoenzyme containing the alternate sigma factor sigma54, regulatory proteins bound at more distally located enhancer sites can activate transcription via DNA looping by taking advantage of the increasing flexibility of DNA over longer distances. While this second mechanism offers a greater possible flexibility in the location of these binding sites, it is not clear how the specificity offered by the proximity of the regulatory protein and the polymerase intrinsic to the first mechanism is maintained. Here we demonstrate that integration host factor (IHF), a protein that induces a sharp bend in DNA, acts both to inhibit DNA-looping-dependent transcriptional activation by an inappropriate enhancer-binding protein and to facilitate similar activation by an appropriate enhancer-binding protein. These opposite effects have the consequence of increasing the specificity of activation of a promoter that is susceptible to regulation by proteins bound to a distal site.

Bacterial Proteins↗

PubMed2001

Quantitative distance information on protein-DNA complexes determined in polyacrylamide gels by fluorescence resonance energy transfer.

In polyacrylamide gels, we have quantitatively determined Forster transfer (fluorescense resonance energy transfer, FRET) between two fluorescent dyes attached to DNA in protein-DNA complexes. The donor-dye fluorescein labeled to DNA retains its free mobility in the polyacrylamide gel, however, its fluorescence properties change. The different quantum yield of fluorescein in the gel is found to be independent of the gel concentration and can thus be quantitatively taken into account by a reduced Forster distance R0 of 46 A compared to 50 A in solution. We have determined global structural properties of two proteins binding to double-labeled DNA using a novel gel-based fluorescence resonance energy transfer assay. In polyacrylamide gels we have analyzed the binding of integration host factor (IHF) and the high mobility group protein NHP6a to their substrate DNA. The measured Forster transfer efficiency allows us to deduce information on the overall shape and the DNA bending angle in the complex.

Acrylic Resins↗

PubMed1991

HU and IHF, two homologous histone-like proteins of Escherichia coli, form different protein-DNA complexes with short DNA fragments.

Using the gel retardation technique we have studied the protein-DNA complexes formed between HU--the major histone-like protein of Escherichia coli--and short DNA fragments. We show that several HU heterodimers bind DNA in a regularly spaced fashion with each heterodimer occupying about 9 base pairs. The alpha 2 and beta 2 HU homodimers form the same structure as the alpha beta heterodimer on double stranded DNA. However when compared to the heterodimer, they bind single stranded DNA with higher affinity. We also show that HU and the Integration Host Factor of E. coli (IHF) form different structures with the same DNA fragments. Moreover, HU seems to enhance the DNA-binding capacity of IHF to a DNA fragment which does not contain its consensus sequence.

Bacterial Proteins↗

PubMed1991

Characterization of the Pseudomonas aeruginosa transposable phage D3112 [corrected] left-end regulatory region.

The nucleotide sequence (2682 bp) of the left end of the Mu-like transposable bacteriophage D3112 cts15 from Pseudomonas aeruginosa was determined. A 720 bp open reading frame (ORF) is located on the bottom strand (positions 892-173), potentially encoding a polypeptide of 240 residues (Mr = 26,329). Specific binding of Escherichia coli Integration Host Factor (IHF) to a site located 907-922 bp from the D3112 left end suggests the existence of a P. aeruginosa IHF and its role, as in Mu, in the regulation of phage development.

Bacterial Proteins↗