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At least 199 records · Page 11Linked to original sources
PubMed1991

Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli.

The glnHPQ operon of Escherichia coli encodes components of the high-affinity glutamine transport system. One of the two promoters of this operon, glnHp2, is responsible for expression of the operon under nitrogen-limiting conditions. The general nitrogen regulatory protein (NRI) binds to two overlapping sites centered at -109 and -122 from the transcription start site and, when phosphorylated, activates transcription of glnHp2 by catalyzing isomerization of the closed sigma 54-RNA polymerase promoter complex to an open complex. The DNA-bending protein integration host factor (IHF) binds to a site immediately upstream of glnHp2 and enhances the activation of open complex formation by NRI phosphate. The NRI-binding sites can be moved several hundred base pairs further upstream without altering the ability of NRI phosphate to activate open complex formation. We propose that the IHF-induced bend can facilitate or obstruct the interaction between NRI phosphate and the closed complex depending on the relative positions of NRI phosphate and sigma 54-RNA polymerase on the DNA.

Bacterial Proteins↗
PubMed1998

Effect of integration host factor of RNA II synthesis in replication of plasmid containing orip 15A.

The synthesis rates of the replication control RNAs of plasmid orip15A. RNA I, an inhibitor of replication, and RNA II, the primer, have been determined using lacZ fusion plasmids, hybridization assay, and reverse transcription polymerase chain reaction (RT-PCR) in Escherichia coli integration host factor-positive (IHF+) and -negative (IHF-) strains containing pACYC184 plasmid (orip15A). In the absence of IHF (E. coli IHF-), expression of the lacZ gene from the PRNAII promoter increased by a factor of 4 compared with the E. coli wild type (IHF+). Also, the increase in expression was more pronounced when the IHF protein was mutated in the ihfB gene than in the ihfA gene. For the PRNAII promoter of oripMB1 (pBR322), no significant differences were found in expression of the lacZ gene in he E. coli strains examined. The level of beta-galactosidase expression from the PRNA promoter of orip 15A shows that the absence of functional IHF in the transformed strains has no effect on expression of the lacZ gene. The synthesis RNA II:RNA I ratio obtained in hybridization assays was 2.4 for E. coli IHF+ and 4.4 for E. coli IHF-. Densitometric analysis of RT-PCR products indicates that the relative levels of RNA I in E. coli IHF+ and IHF-, are equal, but the relative level of RNA II in E. coli IHF is about four times higher than in E. coli IHF+. These results indicate that the IHF protein inhibits transcription from the PRNAII promoter of orip15A plasmid.

Artificial Gene Fusion↗
PubMed1988

Binding of integration host factor (IHF) to the ilvGp1 promoter of the ilvGMEDA operon of Escherichia coli K12.

Crude extracts of Escherichia coli K-12 were found to bind DNA restriction fragments containing ilvGp1. Our analysis using a series of restriction fragments and a BamHI linker mutation indicate that a factor binds to ilvGp1 or adjacent to it. Analysis with mutant strains of E. coli K-12 and purified IHF indicate that IHF binds to ilvGp1. Furthermore, both analysis in vivo and in vitro indicate that IHF precludes transcription from ilvGp1.

Bacterial Proteins↗
PubMed1995

Glucose starvation stimulon of Escherichia coli: role of integration host factor in starvation survival and growth phase-dependent protein synthesis.

The DNA-binding protein IHF was found to be required for starvation survival and for the induction of 14 proteins of the glucose starvation stimulon. Many of these proteins have been shown previously to be general responders to diverse stress conditions. Overexpression of IHF during balanced growth was not sufficient to induce these proteins, but it resulted in an increased synthesis of rpoH-dependent heat shock proteins.

Bacterial Proteins↗
PubMed1992

Supercoiling, integration host factor, and a dual promoter system, participate in the control of the bacteriophage lambda pL promoter.

The high level of efficiency of the bacteriophage lambda pL promoter is dependent upon the topological state of the promoter DNA and the binding of a DNA-bending protein, IHF, to a site centered -86 base-pairs upstream from the pL transcription start site. Abortive initiation assays indicate that DNA supercoiling stimulates open complex formation, whereas IHF enhances promoter recognition. IHF stimulates promoter recognition to the same extent on linear and supercoiled templates. We found that the pL region contains a second promoter, pL2, that initiates transcription 42 base-pairs upstream from pL. Although competitive with pL and inhibited by IHF, mutations in pL2 do not affect the regulation of pL. Stimulation by IHF is helix-face-dependent. IHF inhibits pL when the IHF binding site is displaced a helical half-turn upstream. The pL sequences protected against DNase I digestion by bound IHF and RNA polymerase do not overlap. However, DNase I-hypersensitive sites appear in the region between the two bound proteins. In addition, IHF enhances RNA polymerase binding to pL. These data suggest that stimulation of pL by IHF involves the interaction of IHF and RNA polymerase to form a loop or otherwise distort the DNA between their binding sites.

Bacterial Proteins↗
PubMed1993

Integration host factor (IHF) applied for partial digestion by restriction endonucleases in large DNA molecules (IARC procedure).

The IHF protein of Escherichia coli was successfully used in IHF-mediated Achilles' Heel Cleavage (IHF-AC) technique (Koob et al., 1988; Kur et al., 1992), and leads to the generation of very rare restriction sites in large DNA molecules. The first step of this procedure is methylation of DNA in the presence of IHF, when the overlapping ihf/restriction sites are protected from methylation, and in the second step the DNA is cut by the cognate restriction enzyme. The aim of the present study is to develop a very exact and reproducible procedure to obtain only a few well-defined cuts with the IHF-pre-treated DNA, depending on the variety of all parameters. This technique (IARC, i.e., IHF-assisted rare cutters) employs the restriction enzyme and only one auxiliary protein (IHF). The advantage of the IARC procedure is that no methylation is required (as opposed to the IHF-AC method). Using the IARC approach, the effects of various IHF concentrations were evaluated on cleaving the activity of the DraI, PacI, PmeI, and SwaI enzymes using DNA of phage lambda or the entire genomic 4.7-Mb DNA of E. coli. At low IHF concentrations only a few cut sites were eliminated by IHF binding, but at high IHF concentration, enzymes were able to cut in only one or several specific sites.

Amino Acid Sequence↗
PubMed2002

Integration host factor is essential for the optimal expression of the styABCD operon in Pseudomonas fluorescens ST.

The StyS/StyR two-component regulatory system of Pseudomonas fluorescens ST controls the expression of the styABCD operon coding for the styrene degradation upper pathway. In a previous work we showed that the promoter of the catabolic operon (PstyA) is induced by styrene and repressed to differing extents by organic acids or carbohydrates. In order to study the mechanisms controlling the expression of this operon, we performed a functional analysis on 5' deletions of PstyA by the use of a promoter-probe system. These studies demonstrated that a palindromic region (sty box), located from nucleotides -52 to -37 with respect to the transcriptional start point is essential for PstyA activity. Moreover, additional regulatory regions involved in the modulation of PstyA activity were found along the promoter sequence. In particular, deletion of a putative StyR binding site, homologous to the 3' half of the sty box and located upstream of this box, resulted in 65% reduction of the induction level of the reporter gene. Additionally, we performed bandshift assays with a DNA probe corresponding to PstyA and protein crude extracts from P. fluorescens ST, using specific DNA fragments as competitors. In these experiments we demonstrated that IHF binds an AT-rich region located upstream of the sty box. On the basis of this finding, coupled with the results obtained with PstyA functional analysis, we suggest that the role of the IHF-mediated DNA bend is to bring closer, in an overlapping position, the upstream StyR putative binding site and the downstream sty box, and that the formed complex enhances transcription.

Bacterial Proteins↗
PubMed1997

Role of upstream activation sequences and integration host factor in transcriptional activation by the constitutively active prokaryotic enhancer-binding protein PspF.

PspF, the transcriptional activator of the pspA operon of Escherichia coli, which belongs to the enhancer binding protein (EBP) family of sigma54 activator proteins, is constitutively active in an in vitro transcription assay. PspF protein, together with RNA polymerase holoenzyme containing sigma54, is required for in vitro transcription from the pspA promoter. EBP proteins are typically subject to regulation either by post-translational modification or interaction of a specific ligand with an N-terminal regulatory domain. However, unlike other members of the EBP family, PspF lacks this domain. pspA is positively regulated by IHF in vitro, and this regulation is dependent on the topology of the DNA; a linear template is much more dependent on IHF than a supercoiled template. EBP binding to upstream activating sequences (UAS) in their target promoters is mediated by the C-terminal domain which contains a helix-turn-helix DNA-binding motif. A mutant PspF protein lacking the C-terminal DNA-binding domain is active in vitro, although at much higher concentrations than the wild-type protein. In vitro transcription from pspA templates missing one or both of the UAS sites is reduced relative to wild-type templates, but is still appreciable; however, IHF acts as a negative regulator of pspA transcription on these mutant templates. Thus, PspF bound to non-specific sequences upstream of the pspA promoter can activate pspA transcription, but this activation is inhibited by IHF. These data, taken together, support the model that a precise promoter geometry is necessary for IHF to positively regulate transcription and that IHF may act to prevent activation from inappropriately spaced upstream sites.

Bacterial Proteins↗
PubMed1985

A phi 80 function inhibitory for growth of lambdoid phage in him mutants of Escherichia coli deficient in integration host factor. I. Genetic analysis of the Rha phenotype.

Bacteriophage phi 80 and lambda-phi 80 hybrid phage of the type lambda (QSR)80, in which the rightmost 10% of the lambda genome is replaced by corresponding phi 80 material, are unable to grow lytically in himA and hip/himD mutants of Escherichia coli K12 at 32 degrees. The genetic element responsible for the growth defect, rha, has been mapped to the (QSR)80 region and was located more precisely by restriction enzyme and DNA heteroduplex analysis of mutations that result in loss of the Rha phenotype. Such an Rha mutant carrying a 1.5-kb deletion beginning 0.58 kb from the right end of the chromosome and extending leftward locates the rha locus at least in part within this region of (QSR)80. In addition, a substitution derivative of lambda (QSR)80 was isolated which does not exhibit the Rha phenotype. In this phage, lambda-80hy95, the right half of the (QSR)80 region is replaced by DNA homologous to the 95-100% segment of lambda. In mixed infections in the himA42 host at 32 degrees, lambda + does not complement lambda (QSR)80 for growth and the burst size of the coinfecting lambda + is reduced in comparison to that in a single infection. Deletion mutants of lambda (QSR)80 that grow normally in himA42 at 32 degrees in single infections are inhibited for growth in mixed infections with lambda (QSR)80. These results suggest the existence of a trans-acting function which inhibits phage growth in the absence of HimA or Hip/HimD function. It is likely that the rha gene either encodes that function or indirectly controls its action.

Bacteriophage lambda↗
PubMed1992

Upstream binding sequences of the XylR activator protein and integration host factor in the xylS gene promoter region of the Pseudomonas TOL plasmid.

The xylR and xylS genes, which encode the positive regulators of the TOL plasmid catabolic pathways, are adjacent genes on the TOL plasmid and are transcribed from divergent promoters. Transcription from the xylS gene promoter, Ps, is positively regulated by effector-activated XylR protein and requires the specific RNA polymerase sigma 54 subunit (RpoN). Deletions and point mutations in the Ps upstream region localized the site of XylR interaction to the region between -133 bp and -207 bp (with respect to the transcriptional start of the xylS messenger), which contains an inverted sequence repeat largely homologous to the motif recognised by XylR in the XylR-regulated 'upper' catabolic pathway promoter, Pu. Gel retardation experiments showed binding of IHF to the Ps promoter region. Corresponding sequences showing good homology to the IHF-binding consensus were identified close to the Ps Promoter (between -35 bp and -47 bp, Ps proximal site) and further upstream overlapping the XylR recognition sequence (Ps distal site). In the latter case IHF recognition motifs were found well conserved on both strands at nearly the same position (between -140 bp and -152 bp on the upper and between -141 bp and -153 bp on the lower strand). Expression from Ps, either under inducing or non-inducing conditions, was, however, only slightly influenced by the absence of IHF in an IHF-deficient mutant and thus activation of Ps, like that of other sigma 54-dependent promoters which are rich in Ts, does not absolutely require IHF protein.

Amino Acid Sequence↗
PubMed2006

Functional coupling between the two active sites during Tn 10 transposition buffers the mutation of sequences critical for DNA hairpin processing.

DNA processing reactions often involve multiple components acting in concert to achieve the desired outcome. However, it is usually difficult to know how the components communicate and cooperate to orchestrate an ordered series of events. We address this question in the context of the Tn 10 transposition reaction, in which the DNA cleavage and joining events occur within a higher-order complex containing a transposase dimer, two transposon ends and the DNA-bending host-factor IHF (Integration Host Factor). Previously it was shown that the complex is asymmetric. The a side consists of an IHF protomer initially immobilized by a DNA-loop, but subsequently used to promote conformational changes required for the cleavage steps. The beta side of the complex was considered to fulfil a more passive role. Here we show that the a side of the complex promotes coupled conformational changes at both transposon ends, while the a and beta sides communicate and cooperate to dominate different phases of the transposition reaction. Together, these effects provide for a robust response to critical changes in the transposon end. These findings also explain the intriguing genetic phenotypes of a series of previously reported Tn10 mutants and have consequences for the evolution of new elements.

Binding Sites↗
PubMed2005

Modulation of the sensitivity of FimB recombination to branched-chain amino acids and alanine in Escherichia coli K-12.

Phase variation of type 1 fimbriae of Escherichia coli requires the site-specific recombination of a short invertible element. Inversion is catalyzed by FimB (switching in either direction) or FimE (inversion mainly from on to off) and is influenced by auxiliary factors integration host factor (IHF) and leucine-responsive regulatory protein (Lrp). These proteins bind to sites (IHF site II and Lrp sites 1 and 2) within the invertible element to stimulate recombination, presumably by bending the DNA to enhance synapses. Interaction of Lrp with a third site (site 3) cooperatively with sites 1 and 2 (termed complex 1) impedes recombination. Inversion is stimulated by the branched-chain amino acids (particularly leucine) and alanine, and according to a current model, the amino acids promote the selective loss of Lrp from site 3 (complex 2). Here we show that the central portion of the fim invertible element, situated between Lrp site 3 and IHF site II, is dispensable for FimB recombination but that this region is also required for full amino acid stimulation of inversion. Further work reveals that the region is likely to contain multiple regulatory elements. Lrp site 3 is shown to bind the regulatory protein with low affinity, and a mutation that enhances binding to this element is found both to diminish the stimulatory effects of IVLA on FimB recombination and to inhibit recombination in the absence of the amino acids. The results obtained emphasize the importance of Lrp site 3 as a control element but also highlight the complexity of the regulatory system that affects this site.

Alanine↗
PubMed1992

A novel method for converting common restriction enzymes into rare cutters: integration host factor-mediated Achilles' cleavage (IHF-AC).

Integration host factor (IHF)-mediated protection against enzymatic methylation at ihf-overlapping sites provides the basis for this novel application of the Achilles' cleavage (AC) technique [Koob et al., Science 241 (1988) 1084-1086] for generating rare natural cleavage sites. When applying IHF-AC to plasmid, phage lambda, Escherichia coli and yeast genomes, only a few of the EcoRI, HinfI, and MboI sites (which overlapped the ihf sites) remained cleavable after prior methylation with the cognate M.EcoRI, M.HinfI, or Dam methyltransferases in the presence of IHF. Thus, IHF-AC essentially converted these enzymes into very rare cutters. The extent of cleavage could be controlled by varying the IHF:DNA ratio and temperature. Moreover, the method permits the genomic location and strength of the ihf sites to be determined.

Bacterial Proteins↗
PubMed1989

Site-specific recombination between cloned attP and attB sites from the Haemophilus influenzae bacteriophage HP1 propagated in recombination-deficient Escherichia coli.

Plasmids were constructed which contain both attP and attB DNA segments derived from the insertion sites of the lysogenic bacteriophage HP1 and its host, Haemophilus influenzae. Similar plasmids containing the two junction segments (attL and attR regions) between the phage genome and the lysogenic host chromosome were also prepared. The formation of recombinant dimer plasmids was observed when attP-attB plasmids were propagated in Escherichia coli HB101 (recA), while plasmids containing the junction segments did not form recombinant dimers. Deletion of the phage DNA segment adjacent to the attP site from the attP-attB constructions eliminated detectable recombination, suggesting that this sequence contains the gene encoding the HP1 integrase. No plasmid recombination was observed in strains of E. coli defective in integration host factor. This suggests that integration host factor is important in the expression or activity of the system which produces the site-specific recombination of sequences derived from HP1 and H. influenzae. Further, it suggests that a protein functionally analogous to E. coli integration host factor may be present in H. influenzae.

Bacteriophages↗