Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Unencapsulated Haemophilus influenzae is the second most common etiologic agent of otitis media in children. H. influenzae requires heme for aerobic growth in vitro and is able to utilize hemoglobin and complexes of heme-hemopexin, heme-albumin, and hemoglobin-haptoglobin and ferritransferrin as sources of iron and heme in vitro. Several of the acquisition mechanisms have been characterized and been shown to be heme repressible in vitro. However, little is known about the expression of heme and/or iron acquisition mechanisms during infections in the middle ear. This study was performed to determine if the genes encoding heme and iron acquisition proteins are transcribed during in vivo growth and to compare these findings with those for samples grown in vitro. Reverse transcriptase PCR (RT-PCR) was used to analyze total RNA fractions derived from in vitro- and in vivo-grown H. influenzae. Genes encoding the transferrin-binding proteins TbpA and TbpB, the 100-kDa hemopexin-binding protein HxuA, and the hemoglobin-binding protein HgpA were transcribed during otitis media. Twelve middle ear fluid samples were analyzed by blind RT-PCR to determine the transcriptional status of these genes in H. influenzae during otitis media. Five isolates had transcripts corresponding to tbpA, tbpB, and hxuA. The presence of hgpA transcripts was variable, depending on the presence of hgpA in the genome of the H. influenzae isolate. Samples without H. influenzae gene transcripts contained other etiologic agents commonly causing otitis media. These data demonstrate that H. influenzae iron and/or heme acquisition genes are transcribed during otitis media and suggest that the microenvironment during acute otitis media starves H. influenzae of heme.
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PMID:Transcription of genes encoding iron and heme acquisition proteins of Haemophilus influenzae during acute otitis media. 935 52

Analysis of the Bacillus subtilis genome sequence revealed two open reading frames, designated sigX and ypuN (now termed rsiX), that are homologous to fecI and fecR, respectively, of Escherichia coli. fecI encodes a sigma 70-type factor that is necessary for transcription of the ferric citrate transport genes fecABCDE. fecR encodes a cytoplasmic transmembrane protein that is required for the induction of fec transport gene transcription by ferric citrate binding to the FecA outer membrane receptor protein. Investigation of the SigX and RsiX activities disclosed that they are not involved in ferric citrate utilization--since ferric citrate did not serve as an iron source for B. subtilis SG64--or in the regulation of any other ferric siderophore transport system tested. Strains deleted for sigX or rsiX displayed no phenotype under aerobic or anoxic conditions. However, cloned sigX complemented an E. coli fecI mutant, and the Fur box upstream of sigX responded to the E. coli iron regulatory protein Fur. The purified SigX protein was required for in vitro transcription of a sigX-containing DNA fragment by the E. coli RNA polymerase core enzyme. Autoregulation of sigX was also found in vivo using a sigX'-lacZ gene fusion. RsiX inhibited SigX activity in vivo and in vitro and stabilized the SigX protein. RsiX was localized in the membrane fraction. When RsiX is present, SigX is found in the membrane fraction; in the absence of RsiX, some SigX is detectable in the cytoplasm. We conclude that SigX is a sigma factor that belongs to the ECF (extracytoplasmic function) sigma 70-factor family. It is not known which promoters are recognized by SigX in B. subtilis. SigX may be involved in the regulation of iron metabolism, as evidenced by its activity in E. coli.
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PMID:SigX of Bacillus subtilis replaces the ECF sigma factor fecI of Escherichia coli and is inhibited by RsiX. 939 39

Escherichia coli contains two differentially regulated aconitase genes, acnA and acnB. Two acnA promoters transcribing from start points located 407 bp (P1acnA) and 50 bp (P2acnA) upstream of the acnA coding region, and one acnB promoter (PacnB) with a start point 95 bp upstream of the acnB coding region, were identified by primer extension analysis. A 2.8 kb acnA monocistronic transcript was detected by Northern blot hybridization, but only in redox-stressed (methyl-viologen-treated) cultures, and a 2.5 kb acnB monocistronic transcript was detected in exponential- but not stationary-phase cultures. These findings are consistent with previous observations that acnA is specifically subject to SoxRS-mediated activation, whereas acnB encodes the major aconitase that is synthesized earlier in the growth cycle than AcnA. Further studies with acn-lacZ gene fusions and a wider range of transcription regulators indicated that acnA expression is initiated by sigma 38 from P1acnA, and from P2acnA it is activated directly or indirectly by CRP, FruR, Fur and SoxRS, and repressed by ArcA and FNR. In contrast, acnB expression is activated by CRP and repressed by ArcA, FruR and Fis from PacnB. Comparable studies with fum-lacZ fusions indicated that transcription of fumC, but not of fumA or fumB, is initiated by RNA polymerase containing sigma 38. It is concluded that AcnB is the major citric acid cycle enzyme, whereas AcnA is an aerobic stationary-phase enzyme that is specifically induced by iron and redox-stress.
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PMID:Transcriptional regulation of the aconitase genes (acnA and acnB) of Escherichia coli. 942 4

The mechanism of transcriptional repression of the aerobactin operon of Escherichia coli by the Fe2+-responsive Fur (ferric uptake regulation) protein has been investigated. In the presence of a divalent metal, such as Mn2+, the Fur protein sequentially occupies two defined sites at the aerobactin promoter region, followed by a looser occupation of upstream DNA sequences. However, binding to the primary target site suffices for the entire repression effect. Comparison of transcription patterns generated with run-off experiments in the presence and absence of heparin showed that access of the RNA polymerase to the principal -35/-10 hexamers of the promoter region was fully prevented by Fur-Mn2+ bound to its primary site. Similarly, promoter-bound RNA polymerase could not be competed out from the DNA even in the presence of a large Fur-Mn2+ excess, although the repressor could immediately bind its target sequence at the region as soon as RNA polymerase moved away from the promoter during transcription. The high affinities of either protein for the promoter produce, in practice, a first-come, first-served effect that helps the system to respond instantly to changes in the iron status of the cells.
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PMID:Metalloregulation in vitro of the aerobactin promoter of Escherichia coli by the Fur (ferric uptake regulation) protein. 942 9

Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase alpha subunit were studied using a set of four mutant alpha subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269). These mutant alpha subunits were conjugated with the cutting reagent iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (Fe-BABE), and the peptide backbone was cleaved at locations near the modified Cys. Analysis of the cleavage sites identified segments within approximately 12 A of the conjugation site. These results show that, for intermolecular cutting, segments of the subunit assembly domain (N-terminal domain) of one subunit and the linker region between N- and C-terminal domains of the other subunit are near each other, and the N-terminal domains of both subunits are in close proximity to one another. Intramolecular cutting however, was observed only within an individual N- or C-terminal domain.
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PMID:Dimeric association of Escherichia coli RNA polymerase alpha subunits, studied by cleavage of single-cysteine alpha subunits conjugated to iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate. 947 62

During tetrapyrrole biosynthesis 5-aminolevulinic acid dehydratase (ALAD) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to form one molecule of the pyrrole derivative porphobilinogen. In Escherichia coli, the enzyme is encoded by the gene hemB. The hemB gene was cloned from Pseudomonas aeruginosa by functional complementation of an E. coli hemB mutant. An open reading frame of 1011 bp encoding a protein of 336 amino acids (M(r) 37,008) was identified. The gene was mapped to SpeI fragment G and DpnI fragment G of the P. aeruginosa chromosome, corresponding to the 10 to 12 min region of the new map or 19 to 22 min interval of the old map. The 5' end of the hemB mRNA was determined and the -10 and -35 regions of a potential sigma 70-dependent promoter were localized. No obvious regulation of the hemB gene by oxygen, nitrate, heme or iron was detected. Alignment of the amino acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg(2+)-dependent. P. aeruginosa hemB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg(2+)-dependent activity was directly demonstrated.
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PMID:Cloning, mapping and functional characterization of the hemB gene of Pseudomonas aeruginosa, which encodes a magnesium-dependent 5-aminolevulinic acid dehydratase. 952 30

The mechanism involved in transcriptional repression of the fepA-fes divergent promoters of Escherichia coli by the Fur (ferric uptake regulation) protein has been examined in vitro. This DNA region includes a suboptimal and single Fur-binding site with two divergent and overlapped -35/-10 hexamers. Comparison of transcription patterns generated with runoff experiments in either the presence or the absence of heparin showed that access of the RNA polymerase to the principal -35/-10 hexamers was fully prevented by Fur-Mn2+ bound to its target site within the divergent promoter region. Neither RNA polymerase bound to the fes and fepA promoters could be displaced by Fur-Mn2+, nor could the bound repressor be outcompeted by an excess of the enzyme. However, the repressor blocked reinitiation as soon as the polymerase moved away from the fes promoter during transcription. The spatial distribution of regulatory elements within the DNA region allowed the simultaneous binding of the RNA polymerase to the fes and fepA promoters and their coordinate regulation regardless of their different transcriptional activities. Comparisons with other iron-regulated systems support a general mechanism for Fur-controlled promoters that implies a direct competition between the polymerase and the regulator for overlapping target sites in the DNA.
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PMID:Coordinated repression in vitro of the divergent fepA-fes promoters of Escherichia coli by the iron uptake regulation (Fur) protein. 957 16

Ferric citrate induces transcription of the ferric citrate transport genes fecABCDE in Escherichia coli by binding to the outer-membrane receptor protein FecA without entering the cell. Replete iron concentrations inhibit transcription of the fec transport system via the iron-loaded Fur repressor. Here we show that the Fur repressor activated by Mn2+ (used instead of Fe2+) binds to the promoter of the regulatory genes fecIR and to the promoter of fecABCDE. DNase I footprint analysis revealed that Mn2+-Fur (50 nM) protected 30 nucleotides of the coding strand and 24 nucleotides of the noncoding strand of the fecIR promoter. Higher amounts of Mn2+-Fur (100 nM) covered 41 nucleotides of the coding strand of the fecIR promoter and 38 nucleotides of the coding strand of the fecA promoter. The corresponding region of the noncoding strand of the fecA promoter was hypersensitive to DNase I. The results of a deletion analysis of the fecA promoter supported the previously assigned -35 and -10 regions and nucleotide position +11 for FecI-RNA polymerase interaction. Induction of fecIR transcription by iron limitation increased fecB-lacZ transcription 3.5-fold, whereas under constitutive fecIR transcription, iron limitation increased fecB-lacZ transcription twofold. The two iron-regulated sites of fec transport gene transcription suggest a fast response to sufficient intracellular iron concentrations by repression of fecABCDE transcription and a slower adaptation as the result of fecIR transcription inhibition.
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PMID:Iron regulates transcription of the Escherichia coli ferric citrate transport genes directly and through the transcription initiation proteins. 957 33

Base-specific interactions between promoter DNA and Escherichia coli RNA polymerase are regulated by a sigma (sigma) protein during transcription initiation. To map spatial relations between evolutionarily conserved regions of the primary sigma (sigma 70) and each DNA strand along the lacUV5 promoter in the transcriptionally active "open" complex, we have used a cysteine-tethered cutting reagent to cleave DNA strands. The chemical nuclease FeBABE [iron (S)-1-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate] was conjugated to single-cysteine mutants of sigma 70 at sites 132C, 376C, 396C, 422C, 496C, 517C, or 581C. After formation of open promoter complexes between lacUV5 DNA and RNA polymerase holoenzymes carrying conjugated sigma 70 subunits, we observed promoter DNA cleavage spanning at least 60 bases, between positions -48 and +12. The results show that sigma 70 region 2.1, otherwise implicated in core enzyme binding, is proximal to the nontemplate strand of lacUV5 DNA between the -10 promoter element and positions as far downstream of the transcription start site as +12. Conserved region 3.2 of sigma 70 is proximal to the template strand near the +1 transcription start site, and region 3.1 is positioned between the lacUV5-10 and -35 promoter elements. We propose a model for the orientation of sigma 70 and DNA in the open complex.
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PMID:Mapping the promoter DNA sites proximal to conserved regions of sigma 70 in an Escherichia coli RNA polymerase-lacUV5 open promoter complex. 960 Oct 26

Anthracycline-derivatives are frequently used chemotherapeutics in treatment of numerous human malignancies. Anthracyclines are known for their complex cytotoxic mechanism involving i) inhibition of enzymes such as topoisomerase II, RNA polymerase, cytochrome c oxidase and others; ii) intercalation into DNA; iii) chelation of iron and generation of reactive oxygen species (ROS); iv) induction of apoptosis. Here, mechanistic aspects for successful cytostasis and for side effects, e.g. cardiomyopathy, are discussed. We emphasize recent developments in anthracycline-mediated apoptosis and focus on a well known representative, doxorubicin (adriamycin, adriblastin). We reflect on the role of oxidative stress and interactions with intracellular signaling pathways.
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PMID:Anthracycline-derived chemotherapeutics in apoptosis and free radical cytotoxicity (Review). 985 55


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