Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The leukotoxin of Mannheimia haemolytica is an important virulence factor that contributes to much of the pathology observed in the lungs of animals with bovine shipping fever pneumonia. We believe that identification of factors that regulate leukotoxin expression may provide insight into M. haemolytica pathogenicity. The DNA sequence upstream of the leukotoxin operon is divergently shared by P(lapT), which transcribes an arginine permease gene. The intergenic region contains several elements that are potential sites for transcriptional modulation of the promoters. We have developed plasmid-borne chloramphenicol acetyltransferase (cat) operon fusions, as well as lktC::cat chromosomal fusions, to study transcription initiation in M. haemolytica. Using these genetic tools, we have identified cis-acting sequences and environmental conditions that modulate transcription of the leukotoxin and lapT promoters. By deletion analysis, promoters were shown to rely on sequences upstream of their -10 and -35 regions for full activity. Direct repeats of the sequence TGT-N(11)-ACA and a static bend region caused by phased adenine tracts were necessary for full activation of P(lkt). A computer-generated model of the promoter's structure shows how DNA bending brings the repeat sequences within close proximity to the P(lkt) RNA polymerase, and we hypothesize that these repeats are a binding site for an activator of leukotoxin transcription. The lktC::cat operon fusion was also used to demonstrate that, like that of other RTX toxins, leukotoxin transcription is environmentally regulated. Roles for iron deprivation and temperature change were identified.
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PMID:Use of operon fusions in Mannheimia haemolytica to identify environmental and cis-acting regulators of leukotoxin transcription. 1155 65

The FNR protein of Escherichia coli regulates target genes in response to anaerobiosis. Environmental oxygen is sensed by the acquisition of oxygen-labile [4Fe-4S] clusters that promote dimerization, DNA binding, and productive interactions with RNA polymerase. Three N-terminal cysteine residues (Cys(20), Cys(23), and Cys(29)) and Cys(122) act as ligands for the FNR iron sulfur clusters. An FNR variant, FNR-C20S, that retains only trace activity in vivo can acquire [4Fe-4S] clusters in vitro that enhance site-specific DNA binding. Second site substitutions in activating regions AR1, AR2, and AR3 restore in vivo activity to FNR-C20S, suggesting that the impairment in FNR-C20S activity is due to a failure to communicate with RNA polymerase effectively. Here we show that FNR-C20S can repress a simple FNR-regulated promoter in vivo and that it can form productive heterodimers with an FNR variant with altered DNA binding specificity, FNR-E209V. Transcription studies with FNR-E209V.FNR-C20S heterodimers indicate that the presence of a miscoordinated iron-sulfur cluster (FNR-C20S) in the downstream (but not the upstream) subunit of the FNR dimer impairs activation from a class II promoter and that this impairment can be overcome by amino acid substitutions known to unmask AR2 or improve AR3 in the affected subunit.
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PMID:Miscoordination of the iron-sulfur clusters of the anaerobic transcription factor, FNR, allows simple repression but not activation. 1170 61

In this work, we characterize genes in Mycobacterium tuberculosis that are regulated by IdeR (iron-dependent regulator), an iron-responsive DNA-binding protein of the DtxR family that has been shown to regulate iron acquisition in Mycobacterium smegmatis. To identify some of the genes that constitute the IdeR regulon, we searched the M. tuberculosis genome for promoter regions containing the consensus IdeR/DxR binding sequence. Genes preceded by IdeR boxes included a set encoding proteins necessary for iron acquisition, such as the biosynthesis of siderophores (mbtA, mbtB, mbtI), aromatic amino acids (pheA, hisE, hisB-like) and others annotated to be involved in the synthesis of iron-storage proteins (bfrA, bfrB). Some putative IdeR-regulated genes identified in this search encoded proteins predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)-like molecules (rv3402c), lipids (acpP) and peptidoglycan (murB). We analysed four promoter regions containing putative IdeR boxes, mbtA-mbtB, mbI, rv3402c and bfrA-bfd, for interaction with IdeR and for iron-dependent expression. Gel retardation experiments and DNase footprinting analyses with purified IdeR showed that IdeR binds to these IdeR boxes in vitro. Analysis of the promoters by primer extension indicated that the IdeR boxes are located near the -10 position of each promoter, suggesting that IdeR acts as a transcriptional repressor by blocking RNA polymerase binding. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) coupled to molecular beacons, we showed that mRNA levels of mbtA, mbtB, mbtI, rv3402c and bfd are induced 14- to 49-fold in cultures of M. tuberculosis starved for iron, whereas mRNA levels of bfrA decreased about threefold. We present evidence that IdeR not only acts as a transcriptional repressor but also functions as an activator of bfrA. Three of the IdeR- and iron-repressed genes, mbtB, mbtI and rv3402c, were induced during M. tuberculosis infection of human THP-1 macrophages.
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PMID:The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages. 1172 47

UhpA, a member of the NarL family of response regulators, activates transcription of the Escherichia coli uhpT gene for the sugar phosphate transporter UhpT in response to extracellular glucose-6-phosphate. UhpA binds with different affinities to adjacent regions in the uhpT promoter, termed the strong-binding (S) region from -80 to -50 and the weak-binding (W) region from -50 to -32. Transcription activation by UhpA is stimulated by the catabolite gene activator protein (CAP)-cyclic AMP complex and depends on the C-terminal domains of the RNA polymerase RpoA and RpoD subunits. Because single-base substitutions in the UhpA-binding region had little effect on promoter activity, nucleotide substitutions in successive 4-bp blocks throughout this region were examined for their effects on promoter activation and UhpA binding. Changes in three of four blocks within the W region substantially impaired the ability of UhpA to bind to this region, to drive expression of a uhpT-lacZ reporter, and to support UhpA-dependent in vitro transcription. These W region variant promoters were strongly stimulated by CAP. Changes in several parts of the S region impaired UhpA binding to both the S and W regions and decreased promoter activity in vivo and in vitro. Thus, binding of UhpA to the W region is crucial for UhpA-dependent activation and depends on occupancy of the S region. None of these substitutions eliminated promoter function. The orientation of UhpA-binding sites was assessed by the affinity cleavage method. The iron chelate FeBABE [iron (S)-1-(p-bromoacetamidobenzyl) EDTA] was covalently attached to engineered cysteine residues near the DNA-binding region in UhpA. Hydroxyl radicals generated by the iron chelate attached at position 187 resulted in DNA strand cleavages in two clusters of sites located in the middle of the S and W regions. These results are consistent with the binding of two dimers of UhpA. Each dimer binds to an inverted repeat of monomer-binding sites with the consensus sequence CCTGRR, where R is A or G, and each is separated by 6 bp. It is likely that members of the NarL family bind to dyad targets, in contrast to the binding of OmpR family response regulators to direct-repeat targets.
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PMID:Mutational scanning and affinity cleavage analysis of UhpA-binding sites in the Escherichia coli uhpT promoter. 1197 97

The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia via the assembly-disassembly of oxygen-labile iron-sulfur clusters. Previous work identified patches of surface-exposed amino acids (designated activating regions 1 and 3 [AR1 and AR3, respectively]) of FNR which allow it to communicate with RNA polymerase (RNAP) and thereby activate transcription. Previously it was thought that FNR lacks a functional activating region 2 (AR2), although selecting for mutations that compensate for defective AR1 or a miscoordinated iron-sulfur cluster can reactivate AR2. Here we show that the substitution of two surface-exposed lysine residues (Lys49 and Lys50) of FNR impaired transcription from class II (FNR box centered at -41.5) but not class I (FNR box centered at -71.5) FNR-dependent promoters. The degree of impairment was greater when a negatively charged residue (Glu) replaced either Lys49 or Lys50 than when uncharged amino acid Ala was substituted. Oriented heterodimers were used to show that only the downstream subunit of the FNR dimer was affected by the Lys-->Ala substitutions at a class II promoter. Site-directed mutagenesis of a negatively charged patch ((162)EEDE(165)) within the N-terminal domain of the RNAP alpha subunit that interacts with the positively charged AR2 of the cyclic AMP receptor protein suggested that Lys49 and Lys50 of FNR interact with this region of the alpha subunit of RNAP. Thus, it was suggested that Lys49 and Lys50 form part of a functional AR2 in FNR.
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PMID:Transcription activation by FNR: evidence for a functional activating region 2. 1237 18

Reverse transcriptase PCR analyses have demonstrated that open reading frames (ORFs) PM0298, PM0299, and PM0300 of the animal pathogen Pasteurella multocida constitute a single transcriptional unit. By cloning and overexpression studies in Escherichia coli cells, the product of ORF PM0300 was shown to bind hemoglobin in vitro; this ORF was therefore designated hgbA. In vitro and in vivo quantitative assays demonstrated that the P. multocida hgbA mutant bound hemoglobin to the same extent as the wild-type strain, although the adsorption kinetics was slightly slower for the hgbA cells. In agreement with this, the virulence of P. multocida hgbA cells was not affected, suggesting that other functional hemoglobin receptor proteins must be present in this organism. On the other hand, P. multocida mutants defective in PM0298 and PM0299 could be isolated only when a plasmid containing an intact copy of the gene was present in the cells, suggesting that these genes are essential for the viability of this bacterial pathogen. By adapting the recombinase-based expression technology in vivo to P. multocida, we also demonstrated that the transcriptional PM0298-PM0299-hgbA unit is iron regulated and that its expression is triggered in the first 2 h following infection in a mouse model. Furthermore, hybridization experiments showed that the hgbA gene is widespread in P. multocida strains regardless of their serotype or the animal from which they were isolated.
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PMID:Characterization of the Pasteurella multocida hgbA gene encoding a hemoglobin-binding protein. 1237 70

Members of the sigma70 family of sigma factors are components of the RNA polymerase holoenzyme that direct bacterial or plastid core RNA polymerase to specific promoter elements that are situated 10 and 35 base-pairs upstream of transcription-initiation points. Members of the sigma70 family also function as contact points for some activator proteins, such as PhoB and lambda(cl), and play a role in the initiation process itself. The primary sigma factor, which is essential for general transcription in exponentially growing cells, is reversibly associated with RNA polymerase and can be replaced by alternative sigma factors that co-ordinately express genes involved in diverse functions, such as stress responses, morphological development and iron uptake. On the basis of gene structure and function, members of the sigma70 family can broadly be divided into four main groups. Sequence alignments of the sigma70 family members reveal that they have four conserved regions, although the highest conservation is found in regions 2 and 4, which are involved in binding to RNA polymerase, recognizing promoters and separating DNA strands (so-called 'DNA melting'). The division of the linear sequence of sigma70 factors into four regions is largely supported by recent structural data indicating that primary sigma factors have three stable domains that incorporate regions 2, 3 and 4. Furthermore, structures of the RNA polymerase holoenzyme have revealed that these domains of sigma70 are spread out across one face of RNA polymerase. These structural data are starting to illuminate the mechanistic role of sigma factors in transcription initiation.
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PMID:The sigma70 family of sigma factors. 1254 Feb 96

Brucella abortus reportedly produces the monocatechol siderophore 2,3-dihydroxybenzoic acid (2,3-DHBA) in response to iron limitation. Nucleotide sequence analysis of the cloned DHBA biosynthesis locus from virulent B. abortus 2308 and genetic complementation of defined Escherichia coli mutants were used to identify the B. abortus genes (designated dhbC, -B, and -A) responsible for synthesis of this siderophore. Reverse transcriptase PCR analysis of total RNA with dhb-specific primers demonstrated that dhbC, -B, and -A are transcribed as components of an operon, together with dhbE, a functional homolog of the Escherichia coli entE gene. Homologs of the E. coli entD and Vibrio cholerae vibH genes were also detected in the flanking regions immediately adjacent to the B. abortus dhbCEBA operon, suggesting that B. abortus has the genetic capacity to produce a more complex 2,3-DHBA-based siderophore. Slot blot hybridization experiments and primer extension analysis showed that transcription of the B. abortus dhbCEBA operon originates from two iron-regulated promoters located upstream of dhbC. Consistent with their iron-dependent regulation, both of the dhbCEBA promoter sequences contain typical consensus Fur-binding motifs. Although previously published studies have shown that 2,3-DHBA production is not required for the establishment and maintenance of chronic spleen infection by B. abortus in mice, experimental infection of pregnant cattle with the B. abortus dhbC mutant BHB1 clearly showed that production of this siderophore is essential for wild-type virulence in the natural ruminant host.
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PMID:Genetic organization and iron-responsive regulation of the Brucella abortus 2,3-dihydroxybenzoic acid biosynthesis operon, a cluster of genes required for wild-type virulence in pregnant cattle. 1265 93

Inherited deficiency of the mitochondrial protein frataxin causes neural and cardiac cell degeneration, and Friedreich's ataxia. Five hypotheses for frataxin's mitochondrial function have been generated, largely from work in non-human cells: iron transporter, iron-sulfur cluster assembler, iron-storage protein, antioxidant and stimulator of oxidative phosphorylation. We analyzed gene expression in three human cell types using microarrays, and identified just 48 transcripts whose expression was significantly frataxin-dependent in at least two cell types. Significant decreases in seven transcripts occurred in the sulfur amino acid (SAA) biosynthetic pathway and the iron-sulfur cluster (ISC) biosynthetic pathway to which it is connected. By contrast, we did not observe a single frataxin-dependent transcript that fits with the other four current hypotheses. Quantitative reverse-transcriptase PCR analysis of ISC-S and rhodanese transcripts confirmed that the expression of these genes involved in ISC metabolism was lower in mutants. Amino acid analysis confirmed the defect in SAA metabolism: homocystine, cysteine, cystathionine and serine were significantly decreased in frataxin-deficient cell extracts and mitochondria. An ISC defect was further confirmed by observing decreases in succinate dehydrogenase and aconitase activities, whose activities require ISCs. The ISC-U scaffold protein was specifically decreased in frataxin-deficient cells, suggesting a role for frataxin in its expression or maintenance, and sodium sulfide partially rescued the oxidant-sensitivity of the FRDA cells. Also, multiple transcripts involved in the Fas/TNF/INF apoptosis pathway were up-regulated in frataxin-deficient cells, consistent with a multi-step mechanism of Friedreich's ataxia pathophysiology, and suggesting alternative possibilities for therapeutic intervention.
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PMID:Decreased expression of genes involved in sulfur amino acid metabolism in frataxin-deficient cells. 1283 93

The Frankia sp. strain ACN14a superoxide dismutase SodF was previously shown to be induced in response to Alnus glutinosa root exudates, and its gene was sequenced. We report here the sequence of the 9-kb genomic segment surrounding the sodF gene and further characterize this gene and its product. Nine ORFs coding for various proteins, such as regulators, acetyl-CoA transferases, and a bacterioferritin A next to the sodF gene, were found. Northern blot analysis showed that the sodF gene was expressed as a major 1-kb transcript, which indicates that it has its own promoter. The sodF gene strongly complemented an Escherichia coli triple mutant (sodA sodB recA), restoring aerobic growth when the gene was expressed from the synthetic tac promoter but when expressed from its own promoter showed only slight rescue, suggesting that it was poorly recognized by the E. coli RNA polymerase. It is noteworthy that this is the first time that a Frankia gene has been reported to complement an E. coli mutant. The superoxide dismutase activity of the protein was inactivated by hydrogen peroxide, indicating that the metal ligand is iron, which is supported by analysis of the protein sequence. Thus, the SodF protein induced in Frankia by root exudates is an iron-containing enzyme similar to the one present in the nodules.
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PMID:Characterization of the sodF gene region of Frankia sp. strain ACN14a and complementation of Escherichia coli sod mutant. 1289 39


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