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)

Environmental and clinical isolates of mercury-resistant (resistant to inorganic mercury salts and organomercurials) bacteria have genes for the enzymes mercuric ion reductase and organomercurial lyase. These genes are often plasmid-encoded, although chromosomally encoded resistance determinants have been occasionally identified. Organomercurial lyase cleaves the C-Hg bond and releases Hg(II) in addition to the appropriate organic compound. Mercuric reductase reduces Hg(II) to Hg(O), which is nontoxic and volatilizes from the medium. Mercuric reductase is a FAD-containing oxidoreductase and requires NAD(P)H and thiol for in vitro activity. The crystal structure of mercuric ion reductase has been partially solved. The primary sequence and the three-dimensional structure of the mercuric reductase are significantly homologous to those of other flavin-containing oxidoreductases, e.g., glutathione reductase and lipoamide dehydrogenase. The active site sequences are the most conserved region among these flavin-containing enzymes. Genes encoding other functions have been identified on all mercury ion resistance determinants studied thus far. All mercury resistance genes are clustered into an operon. Hg(II) is transported into the cell by the products of one to three genes encoded on the resistance determinants. The expression of the operon is regulated and is inducible by Hg(II). In some systems, the operon is inducible by both Hg(II) and some organomercurials. In gram-negative bacteria, two regulatory genes (merR and merD) were identified. The (merR) regulatory gene is transcribed divergently from the other genes in gram-negative bacteria. The product of merR represses operon expression in the absence of the inducers and activates transcription in the presence of the inducers. The product of merD coregulates (modulates) the expression of the operon. Both merR and merD gene products bind to the same operator DNA. The primary sequence of the promoter for the polycistronic mer operon is not ideal for efficient transcription by the RNA polymerase. The -10 and -35 sequences are separated by 19 (gram-negative systems) or 20 (gram-positive systems) nucleotides, 2 or 3 nucleotides longer than the 17-nucleotide optimum distance for binding and efficient transcription by the Escherichia coli sigma 70-containing RNA polymerase. The binding site of MerR is not altered by the presence of Hg(II) (inducer). Experimental data suggest that the MerR-Hg(II) complex alters the local structure of the promoter region, facilitating initiation of transcription of the mer operon by the RNA polymerase. In gram-positive bacteria MerR also positively regulates expression of the mer operon in the presence of Hg(II).
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PMID:Bacterial resistances to inorganic mercury salts and organomercurials. 131 Nov 13

The Escherichia coli gene murB, encoding the enzyme uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucosenicoti namide adenine dinucleotide phosphate oxidoreductase (EC 1.1.1.158) (EP-reductase), the second enzyme in the peptidoglycan biosynthetic pathway, has been amplified using PCR technology with the Kohara recombinant lambda phage E11C11 (534) as template. The synthetic gene was subcloned into the NdeI and BamHI restriction sites of the expression vector pT7-7, designed to utilize T7 RNA polymerase to direct transcription of the target gene, in a two-step procedure. The first step involved the directional insertion of the 590-bp NdeI to BamHI restriction fragment of murB into the pT7-7 vector to give the plasmid pT7-7-murB-590. The construction of the desired overproducing plasmid was completed by the bidirectional insertion of the 442-bp BamHI to BamHI restriction fragment of murB into a similarly restricted pT7-7-murB-590 plasmid followed by restriction digestion to select the properly oriented insert, pT7-7-murB. Overexpression of EP-reductase from the E. coli strain BL 21 (DE 3) containing the pT7-7-murB gene, after induction, allowed the production of 36 mg of target protein per 3 wet grams of E. coli cells. The EP-reductase was purified in a single step utilizing dye-ligand chromatography to yield 30 mg of pure protein. The availability of these levels of reductase will allow the mechanism of this pivotal enzyme to be thoroughly studied as a potential target for the design of a new generation of antibiotics. In addition, the EP-reductase generated in this study has been utilized as a coupling enzyme to assay the first enzyme in the peptidoglycan biosynthetic pathway, UDP-N-acetylglucosamine enolpyruvyl transferase, and these results are also presented.
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PMID:Overproduction and one-step purification of Escherichia coli UDP-N-acetylglucosamine enolpyruvyl reductase. 874 27

The findings presented here originally arose from the suggestion that the synthesis of dinucleoside polyphosphates (Np(n)N) may be a general process involving enzyme ligases catalyzing the transfer of a nucleotidyl moiety via nucleotidyl-containing intermediates, with release of pyrophosphate. Within this context, the characteristics of the following enzymes are presented. Firefly luciferase (EC 1.12. 13.7), an oxidoreductase with characteristics of a ligase, synthesizes a variety of (di)nucleoside polyphosphates with four or more inner phosphates. The discrepancy between the kinetics of light production and that of Np(n)N synthesis led to the finding that E*L-AMP (L = dehydroluciferin), formed from the E*LH(2)-AMP complex (LH(2) = luciferin) shortly after the onset of the reaction, was the main intermediate in the synthesis of (di)nucleoside polyphosphates. Acetyl-CoA synthetase (EC 6.2.1.1) and acyl-CoA synthetase (EC 6.2.1. 8) are ligases that synthesize p(4)A from ATP and P(3) and, to a lesser extent, Np(n)N. T4 DNA ligase (EC 6.5.1.1) and T4 RNA ligase (EC 6.5.1.3) catalyze the synthesis of Np(n)N through the formation of an E-AMP complex with liberation of pyrophosphate. DNA is an inhibitor of the synthesis of Np(n)N and conversely, P(3) or nucleoside triphosphates inhibit the ligation of a single-strand break in duplex DNA catalyzed by T4 DNA ligase, which could have therapeutic implications. The synthesis of Np(n)N catalyzed by T4 RNA ligase is inhibited by nucleoside 3'(2'),5'-bisphosphates. Reverse transcriptase (EC 2.7.7.49), although not a ligase, catalyzes, as reported by others, the synthesis of Np(n)ddN in the process of removing a chain termination residue at the 3'-OH end of a growing DNA chain.
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PMID:Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases. 1100 93

Studies were carried out to elucidate the mechanism whereby thyroid hormone (T3) induces NADPH:cytochrome P450 oxidoreductase (P450R) mRNA in rat liver in vivo. Northern blot analysis revealed that T3 treatment increases unspliced liver nuclear P450R RNA 4-fold within 8 h and that this induction precedes the induction of mature, cytoplasmic P450R RNA. Unspliced nuclear P450R RNA was suppressed below basal levels 24 h after T3 treatment, despite the continued presence of elevated circulating T3 levels. To determine whether the T3-stimulated increase in nuclear P450R RNA reflects an increase in P450R transcription initiation, nuclear run-on transcription assays were carried out. T3 induced a 6- to 8-fold increase in P450R transcription rate within 12 h, sufficient to account for the observed increase in nuclear P450R precursor RNA, followed by a decrease back to basal transcription levels at 24 h, consistent with the nuclear RNA profile. Similar transcriptional increases were observed in nuclear run-on transcription studies using hybridization probes corresponding to nine different fragments of the P450R gene, spanning exon 2 to exon 16. Thus, P450R transcription initiation, not transcription elongation, is the T3-regulated event. Similar results were obtained during short (5 min) compared with long (45 min) nuclear run-on transcription assays, suggesting that changes in nuclear RNA processing or regulated degradation do not contribute to the overall RNA induction. This finding was confirmed by the ability of the RNA polymerase inhibitor actinomycin D, administered in vivo, to block T3 induction of P450R transcriptional activity. We conclude that P450R transcription, rather than nuclear RNA processing or mRNA stabilization, is the primary mechanism whereby T3 induces hepatic P450R mRNA.
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PMID:Transcriptional induction of hepatic NADPH: cytochrome P450 oxidoreductase by thyroid hormone. 1130 80

A cDNA encoding a new cytochrome P450 was isolated from a mouse brain library. Sequence analysis reveals that this 1,958-base pair cDNA encodes a 57-58-kDa 502-amino acid polypeptide that is 70-91% identical to CYP2J subfamily P450s and is designated CYP2J9. Recombinant CYP2J9 was co-expressed with NADPH-cytochrome P450 oxidoreductase (CYPOR) in Sf9 cells using a baculovirus system. Microsomes of CYP2J9/CYPOR-transfected cells metabolize arachidonic acid to 19-hydroxyeicosatetraenoic acid (HETE) thus CYP2J9 is enzymologically distinct from other P450s. Northern analysis reveals that CYP2J9 transcripts are present at high levels in mouse brain. Mouse brain microsomes biosynthesize 19-HETE. RNA polymerase chain reaction analysis demonstrates that CYP2J9 mRNAs are widely distributed in brain and most abundant in the cerebellum. Immunoblotting using an antibody raised against human CYP2J2 that cross-reacts with CYP2J9 detects a 56-kDa protein band that is expressed in cerebellum and other brain segments and is regulated during postnatal development. In situ hybridization of mouse brain sections with a CYP2J9-specific riboprobe and immunohistochemical staining with the anti-human CYP2J2 IgG reveals abundant CYP2J9 mRNA and protein in cerebellar Purkinje cells. Importantly, 19-HETE inhibits the activity of recombinant P/Q-type Ca(2+) channels that are known to be expressed preferentially in cerebellar Purkinje cells and are involved in triggering neurotransmitter release. Based on these data, we conclude that CYP2J9 is a developmentally regulated P450 that is abundant in brain, localized to cerebellar Purkinje cells, and active in the biosynthesis of 19-HETE, an eicosanoid that inhibits activity of P/Q-type Ca(2+) channels. We postulate that CYP2J9 arachidonic acid products play important functional roles in the brain.
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PMID:Cytochrome P450 CYP2J9, a new mouse arachidonic acid omega-1 hydroxylase predominantly expressed in brain. 1132 10

One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalyzed conversion into 3-dehydroecdysteroid followed by irreversible reduction by 3-dehydroecdysone 3alpha-reductase to 3-epiecdysone. We have purified from Spodoptera littoralis the first ecdysone oxidase and subjected it to limited amino acid sequencing. A reverse-transcriptase polymerase chain reaction-based approach has been used to clone the cDNA (2.8 kilobases) encoding this 65-kDa protein. Northern blotting showed that the mRNA transcript was expressed in midgut during the prepupal stage of the last larval instar at a time corresponding to an ecdysteroid titer peak. Conceptual translation of the ecdysone oxidase cDNA and data base searching revealed that the enzyme is an FAD flavoprotein that belongs to the glucose-methanol-choline oxidoreductase superfamily. Ecdysone oxidase represents the only oxidase in eukaryotic animals known to catalyze oxygen-dependent oxidation of steroids; by contrast, oxidation of steroids in vertebrates occurs via NAD(P)(+)-linked dehydrogenases. The injection of RH-5992, an ecdysteroid agonist, induced the transcription of ecdysone oxidase, suggesting that ecdysone oxidase is an ecdysteroid-responsive gene. The gene encoding this enzyme, consisting of five exons, has also been isolated. Sequences similar to the binding motifs for Broad-Complex and FTZ-F1 have been found in the 5'-flanking region. Southern blotting indicated that ecdysone oxidase is encoded by a single-copy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system.
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PMID:Regulation of ecdysteroid signaling: cloning and characterization of ecdysone oxidase: a novel steroid oxidase from the cotton leafworm, Spodoptera littoralis. 1137 99

The present study evaluates the expression of genes of Giardia lamblia, one of the most simple and most early diverging eukaryotes, that encode the metabolic enzymes pyruvate: ferredoxin oxidoreductase (PFOR), acetyl-CoA synthetase (ACS), alcohol dehydrogenase E (ADHE) and glutamate dehydrogenase (GDH) and the cyst wall protein (CWP1) gene in trophozoites, cysts and during the excystation process. Primers were designed to amplify mRNA fragments through quantitative reverse-transcriptase-polymerase-chain-reaction. In trophozoites, all transcripts of the enzymes studied were present. In cysts, three of the transcripts were detected: CWP1, GDH and ACS; but the relative levels of the mRNA of GDH and ACS were very different between trophozoites and cysts. During excystation, PFOR and ADHE transcripts appeared after the first induction phase, and the mRNAs of ACS and GDH increased throughout the process.
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PMID:Transcription of metabolic enzyme genes during the excystation of Giardia lamblia. 1466 85

Nitric oxide is an intermediate of denitrification, and is one of the radical species deployed by macrophages against invading pathogens, therefore bacterial responses to NO are of considerable importance. The Escherichia coli flavorubredoxin and its associated oxidoreductase reduce NO to nitrous oxide under anaerobic conditions, and are encoded by the norVW transcription unit. Expression of norVW requires the NO sensing regulatory protein NorR and is dependent on RNA polymerase containing the alternative sigma factor, sigma(54). We have purified NorR and shown that it binds to three sites in the norVW promoter region, located 75-140 bp upstream of the experimentally verified transcription start site. We have also identified two binding sites for the integration host factor, one between the NorR sites and the sigma(54)-RNA polymerase binding site, and a second downstream of the norVW transcription start site. Comparison of the norVW promoters of enteric bacteria along with known and putative NorR-regulated promoters from Vibrio, Ralstonia and Pseudomonas species suggests that NorR binding sites contain an invariant GT(N7)AC motif flanking an AT-rich central region. The identification of a consensus for NorR binding sites will help to elucidate additional members of the NorR regulon.
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PMID:DNA binding properties of the Escherichia coli nitric oxide sensor NorR: towards an understanding of the regulation of flavorubredoxin expression. 1566

Carotenoids are produced by a variety of organisms, but the mechanisms that regulate gene expression leading to carotenoid biosynthesis have been characterized for only a few organisms. In this study, we found that Streptomyces coelicolor A3(2), a gram-positive filamentous bacterium, produces carotenoids under blue light induction. The carotenoid fraction isolated from the cell extract contained multiple compounds, including isorenieratene and beta-carotene. The carotenoid biosynthesis gene cluster of S. coelicolor consists of two convergent operons, crtEIBV and crtYTU, as previously shown for Streptomyces griseus. The crtEIBV null mutant completely lost its ability to produce carotenoids. The crt gene cluster is flanked by a regulatory region that consists of two divergent operons, litRQ and litSAB. The lit (light-induced transcription) genes encode a MerR-type transcriptional regulator (LitR), a possible oxidoreductase (LitQ), an extracytoplasmic function sigma factor (sigmaLitS), a putative lipoprotein (LitA), and a putative anti-sigma factor (LitB). S1 protection assay revealed that the promoters preceding crtE (PcrtE), crtY (PcrtY), litR (PlitR), and litS (PlitS) are activated upon illumination. A litS mutant lost both the ability to produce carotenoids and the activities of PcrtE, PcrtY, and PlitS, which suggested that sigmaLitS directs light-induced transcription from these promoters. An RNA polymerase holocomplex containing purified sigmaLitS recombinant protein generated specific PcrtE and PcrtY transcripts in an in vitro runoff transcriptional assay. A litR mutant that had an insertion of the kanamycin resistance gene was defective both in the ability to produce carotenoids and in all of the light-dependent promoter activities. Overexpression of litS resulted in constitutive carotenoid production in both the wild type and the litR mutant. These results indicate that sigmaLitS acts as a light-induced sigma factor that directs transcription of the crt biosynthesis gene cluster, whose activity is controlled by an unknown LitR function. This is the first report to describe light-inducible gene expression in Streptomyces.
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PMID:Light-induced carotenogenesis in Streptomyces coelicolor A3(2): identification of an extracytoplasmic function sigma factor that directs photodependent transcription of the carotenoid biosynthesis gene cluster. 1571 54

In this study, we cloned and characterized a human gene homologous to the apoptosis-inducing factor (AIF), which is named AIF-like (AIFL). Human AIFL has 598 amino acids, with a characteristic Rieske domain and a pyridine nucleotide-disulfide oxidoreductase domain (Pyr_redox). AIFL shares 35% homology with AIF, mainly in the Pyr_redox domain. Reverse transcriptase-PCR analysis showed the expression of AIFL mRNA in all tissues tested, i.e. brain, colon, heart, kidney, liver, lung, muscle, ovary, pancreas, placenta, small intestine, and testis. We developed antibodies against human AIFL using fusion proteins as antigens. The antibodies specifically recognized the antigen and heterologously expressed AIFL proteins. The expression of AIFL proteins in human tissues was also ubiquitous, demonstrated by immunohistochemistry in tissue array slides. Subcellular fractionation and immunofluorescence staining studies revealed that AIFL is predominantly localized to the mitochondria. Similar to AIF, overexpression of AIFL induced apoptosis, as shown by increased cytoplasmic nucleosomes and subdiploid cell populations in AIFL-transfected cells. The segment 1-190 containing the Rieske domain induced apoptosis, whereas the segment containing the Pyr_redox domain did not contribute to the pro-apoptotic function. The mitochondrial membrane potential of cells transfected with AIFL was significantly more depolarized than that of the control. AIFL transfection-induced cytochrome c release and cleavage of caspase 3. Furthermore, the pan-caspase inhibitor Z-VAD-fmk inhibited AIFL induced apoptosis. In summary, AIFL induces apoptosis in a caspase-dependent manner when heterologously expressed.
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PMID:Molecular cloning and characterization of a human AIF-like gene with ability to induce apoptosis. 1576 4


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