The HimA and HimD subunits of integration host factor can specifically bind to DNA as homodimers.

Zulianello, Laurence; Rosny, Eve de; Ulsen, Peter van; Putte, Pieter van de; Goosen, Nora

doi:10.1002/j.1460-2075.1994.tb06415.x

Cited by 53 publications

(38 citation statements)

References 32 publications

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“…However, since this derepression is only 1.7-fold, it is possible that in the absence of the himAencoded ␣-subunit for the formation of himA-himD encoded ␣␤-heterodimers, himD-encoded ␤␤-homodimers can function to autoregulate himD expression. Indeed, several examples of the in vivo formation of ␤␤-homodimers functionally competent to recognize and bind to natural IHF-binding sites have been reported (43)(44)(45)(46).…”

Section: Examples Of Direct Effects Of Ihf On Gene Expression Profilementioning

confidence: 99%

Global Gene Expression Profiling in Escherichia coliK12

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Journal of Biological Chemistry

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We have used nylon membranes spotted in duplicate with full-length polymerase chain reaction-generated products of each of the 4,290 predicted Escherichia coli K12 open reading frames (ORFs) to measure the gene expression profiles in otherwise isogenic integration host factor IHF ؉ and IHF ؊ strains. Our results demonstrate that random hexamer rather than 3 ORF-specific priming of cDNA probe synthesis is required for accurate measurement of gene expression levels in bacteria. This is explained by the fact that the currently available set of 4,290 unique 3 ORF-specific primers do not hybridize to each ORF with equal efficiency and by the fact that widely differing degradation rates (steady-state levels) are observed for the 25-base pair region of each message complementary to each ORF-specific primer. To evaluate the DNA microarray data reported here, we used a linear analysis of variance (ANOVA) model appropriate for our experimental design. These statistical methods allowed us to identify and appropriately correct for experimental variables that affect the reproducibility and accuracy of DNA microarray measurements and allowed us to determine the statistical significance of gene expression differences between our IHF ؉ and IHF ؊ strains. Our results demonstrate that small differences in gene expression levels can be accurately measured and that the significance of differential gene expression measurements cannot be assessed simply by the magnitude of the fold difference. Our statistical criteria, supported by excellent agreement between previously determined effects of IHF on gene expression and the results reported here, have allowed us to identify new genes regulated by IHF with a high degree of confidence.

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Section: Examples Of Direct Effects Of Ihf On Gene Expression Profilementioning

confidence: 99%

Global Gene Expression Profiling in Escherichia coliK12

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et al. 2000

Journal of Biological Chemistry

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“…Yang and Nash (37) proposed a model for IHF interaction with DNA in which the "arms" of the IHF protein contact DNA through the minor groove; the C termini provide for additional contacts made possible by the bending of the DNA. Although the two subunits of IHF are similar in structure, experimental evidence indicates that the two subunits contribute differently to the function of IHF (34,38).…”

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Structure and function of the Pseudomonas putida integration host factor

et al. 1996

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Integration host factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P U promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.Integration host factor (IHF) is a sequence-specific DNAbinding and -bending heterodimeric protein that is abundant in the Escherichia coli cell. Its two subunits are coded by the ihfA gene (previously termed himA) and by the ihfB gene (previously termed hip or himD) (33). IHF belongs to the family of bacterial histone-like proteins that share amino acid sequence homology with HU proteins from a variety of bacteria (3,5,7,23). One member of the family, HU of Bacillus stearothermophilus, was crystallized (28, 35), and its three-dimensional structure serves as a model for the whole family. According to the model, the N-terminal half of the protein is involved in dimer formation, and the long arms, consisting of antiparallel double-stranded ␤ ribbons of the two subunits, wrap around the DNA. Recent solution structure studies of HU showed that its arms are highly flexible (32).IHF participates in a number of cellular processes such as site-specific recombination, transposition and inversion, phage DNA packaging, plasmid and phage DNA replication, and the positive and negative control of gene expression (for reviews, see references 6 and 7). Yang and Nash (37) proposed a model for IHF interaction with DNA in which the "arms" of the IHF protein contact DNA through the minor groove; the C termini provide for additional contacts made possible by the bending of the DNA. Although the two subunits of IHF are similar in structure, experimental evidence indicates that the two subunits contribute differently to the function of IHF (34, 38).IHF was found to participate in gene regulation in a number of gram-negative bacteria. A large number of these genes are transcribed by ...

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“…It is possible that functional himD homodimers were formed in the himA mutant used for the microarray assay, resulting in activation of the gltBDF operon transcription to a certain extent (3,38).…”

Section: Discussionmentioning

confidence: 99%

Activation from a Distance: Roles of Lrp and Integration Host Factor in Transcriptional Activation of gltBDF

2001

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The leucine-responsive regulatory protein (Lrp) binds to three sites centered 252, 216, and 152 bp upstream of the transcription start site of the Escherichia coli glutamate synthase operon (gltBDF) and activates transcription. Activators of 70 -dependent promoters usually bind closer to the ؊35 hexamer of the core promoter sequence. To study the mechanism by which Lrp-dependent activation occurs over this relatively large distance, the gltBDF upstream region was sequentially replaced with corresponding portions from the well-characterized 70 -dependent promoter lacZYAp. The glt-lac promoter hybrids were placed upstream of lacZ, allowing transcriptional activity to be monitored via ␤-galactosidase assays. Even replacing all gltBDF sequences downstream of and including the ؊35 hexamer did not eliminate Lrp-dependent activation of transcription. When a 91-bp region between the ؊35 hexamer and the proximal Lrp binding site (؊48 to ؊128) was replaced with heterologous DNA of the same length, transcription was reduced nearly 40-fold. Based on the presence of a consensus binding sequence, this region seemed likely to be a binding site for integration host factor (IHF). Experiments to study the effects of a himD mutant on expression of a gltB::lacZ transcriptional fusion, gel mobility shift analyses, and DNA footprinting assays were used to confirm the direct participation of IHF in gltBDF promoter regulation. Based on these results, we suggest that IHF plays a crucial architectural role, bringing the distant Lrp complex in close proximity to the promoter-bound RNA polymerase.Escherichia coli synthesizes glutamate under ammonia-limiting conditions using the enzymes glutamate synthase, specified by the gltBDF operon, and glutamine synthetase, specified by glnA. The genes gltB and gltD specify the large and small subunits of glutamate synthase, respectively (7), and gltF specifies a protein of unknown function (8,20). The gltBDF operon is positively regulated by the leucine-responsive regulatory protein (Lrp) (12,13) and is transcribed at very low levels in the absence of Lrp (12). The coregulator for Lrp is leucine, and exogenous leucine reduces the expression of gltBDF about twofold, although this level is still substantially above baseline transcription in the absence of Lrp. Thus, relative to more responsive operons, gltBDF is a leucine-independent member of the Lrp regulon (12).There are three Lrp binding sites centered 246, 215, and 152 bp upstream of the gltBDF transcription start site (35); the closest of these sites is ϳ110 bp from the Ϫ35 hexamer. The presence of such distal regulatory sites is unusual in 70 -regulated promoters, although it has precedence in promoters that are recognized by 54 (6, 9, 28). In 54 -dependent promoters, upstream elements or activators binding far upstream of the core promoter sequence are brought closer to the RNA polymerase by a looping mechanism that is sometimes assisted by DNA-bending proteins (9), raising the question of whether additional regulatory proteins are required for...

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The HimA and HimD subunits of integration host factor can specifically bind to DNA as homodimers.

Cited by 53 publications

References 32 publications

Global Gene Expression Profiling in Escherichia coliK12

Global Gene Expression Profiling in Escherichia coliK12

Structure and function of the Pseudomonas putida integration host factor

Activation from a Distance: Roles of Lrp and Integration Host Factor in Transcriptional Activation of gltBDF

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