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)

Phage T7 DNA polymerase consists of a 1:1 complex of the viral T7 gene 5 protein and the host cell thioredoxin. A 3.25-kilobase T7 DNA fragment containing the complete coding sequence of gene 5, and the nearby genes 4.7 and 5.3, was cloned in the BamHI site of the plasmid pBR322. Transformation of the thioredoxin-negative (trxA-) Escherichia coli strain BH215 with the recombinant plasmid pRS101 resulted in large overproduction of gene 5 protein corresponding to a level about 60-fold higher than in T7-infected cells. Transcription of gene 5 probably originates from a previously unknown E. coli RNA polymerase promoter located immediately upstream of the structural gene. Contrary to expectation, pRS101 could be maintained also in E. coli trxA+ cells despite the in vivo formation of active T7 DNA polymerase. However, the expression of gene 5 was lower by a factor of 5-10 than in trxA- cells. Since the plasmid copy number in the two strains was the same, a gene dosage effect can be excluded. The observed difference suggests an autoregulatory interaction of T7 DNA polymerase holoenzyme on the expression of T7 gene 5. The trxA- strain BH215/pRS101 is an excellent source of gene 5 protein and T7 DNA polymerase. After in vitro reconstitution of holoenzyme by addition of excess thioredoxin, highly active T7 DNA polymerase was purified to homogeneity by a simple antithioredoxin immunoadsorbent chromatography technique.
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PMID:Bacteriophage T7 DNA polymerase: cloning and high-level expression. 299 84

The virion-associated RNA polymerase and the structural nucleoprotein of influenza A virus were separated by sodium dodecyl sulfate/PAGE, electroblotted to a polyvinylidine membrane, and eluted with good recovery from the membrane. After renaturation by incubating with Escherichia coli thioredoxin, these proteins were active in a reconstituted in vitro transcription reaction with purified genomic RNAs. All four proteins (i.e., the three subunits of the RNA polymerase as well as the structural nucleoprotein) were required for activity. The RNA products were plus-strand, mRNA-sized species.
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PMID:Purification, thioredoxin renaturation, and reconstituted activity of the three subunits of the influenza A virus RNA polymerase. 305 75

Salmonella enterotoxin (Stn) is a virulence factor in S. typhimurium strain Q1 that causes both fluid secretion in ligated intestinal loops of rabbits and elongation of Chinese hamster ovary (CHO) cells. High-level expression systems are needed to provide Stn in soluble form for detailed study of the biological activity of Stn. To maximize the synthesis and solubility of Stn, we systematically compared the production of native Stn synthesized with a T7 RNA polymerase/promoter system to that of two fusion proteins: glutathione S-transferase::Stn (Gst::Stn) and thioredoxin A::Stn (TrxA::Stn). The latter fusion protein expression systems resulted in a 64-fold increase in Gst::Stn and TrxA::Stn antigen concentration, as measured by specific anti-peptide antibodies in an enzyme-linked immunosorbent assay (ELISA). Most of the toxin derived using these vector systems was insoluble; however, the solubility of the TrxA::Stn antigen increased by at least 50-fold, with a concomitant increase in CHO cell elongation activity. In addition, stn gene expression was enhanced more than 50-fold by addition of 0.2-0.4 M NaCl to Luria-Bertani medium. The biological activity of Stn also was increased in the high-osmolarity medium. Consequently, the expression of stn may be regulated by DNA supercoiling.
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PMID:Improved synthesis of Salmonella typhimurium enterotoxin using gene fusion expression systems. 802 62

A human thioredoxin cDNA was modified to optimize Escherichia coli expression and subcloned into the plasmid pACA, a vector for T7 RNA polymerase-directed expression. The substitution of structural (noncatalytic) half-cystines in human thioredoxin (hTrx) was made by site-directed mutagenesis. The recombinant wild-type (wt) hTrx and its mutant C61S, C72S, and C61S/C72S were expressed and purified to homogeneity. Characterization of the wt and mutant hTrx was done with respect to redox activity with thioredoxin reductase (TR), tryptophan fluorescence, and effects of incubation with GS-Se-SG, which is believed to be the major metabolite of inorganic selenium compounds in mammalian tissues. The Km and kcat of wild-type hTrx for human placenta thioredoxin reductase (HP-TR) at pH 7.0 were 2.0 microM and 2800 min-1, respectively. The mutant proteins C61S, C72S, and C61S/C72S had Km and kcat values similar to those of the wt thioredoxin. Tryptophan fluorescence measurements showed that the wt and mutant proteins had similar stability to a denaturing agent. Incubation of fully reduced thioredoxin with 0.1 molar equivalent of GS-Se-SG resulted in continued oxidation of SH groups. After 3.5 h only 0.5 of initially 4.6 SH groups/thioredoxin remained. With the oxidized protein, a pronounced lag phase in thioredoxin reductase-dependent insulin disulfide reduction was present. Disulfide-linked dimers of the protein were present. The results clearly showed that noncatalytic cysteine residues in hTrx were oxidized accompanied by dimerization and inactivation. The activities of the mutant proteins C72S and C61S/C72S were unchanged after 3 h of incubation with GS-Se-SG. No dimer appeared of the C72S thioredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mutagenesis of structural half-cystine residues in human thioredoxin and effects on the regulation of activity by selenodiglutathione. 837 74

The nucleotide sequence relatedness between the chromosome of Salmonella typhi and the virulence plasmid of Salmonella enteritidis was investigated using short DNA probes of < 2 kb covering the whole virulence plasmid sequence. Only one homologous region was detected. This region was subsequently cloned and partially sequenced. Sequences closely related to the pefl gene and the ORFs orf7, orf8 and orf9, which are located downstream of the fimbrial pef operon of the Salmonella typhimurium virulence plasmid, were detected. Sequencing of the cloned S. typhi DNA fragment also revealed identity with genes of the fimbrial sef operon characterized in the chromosome of S. enteritidis. These nucleotide sequences mapped upstream of the S. typhi chromosomal region homologous to the S. enteritidis virulence plasmid. The general organization of the cloned S. typhi chromosomal fragment was similar to the fimbriae-encoding region of the S. typhimurium virulence plasmid. The deduced product of orf8 in the S. typhimurium virulence plasmid, as well as those of the corresponding ORFs in the homologous region of the S. typhi chromosome and in the S. enteritidis virulence plasmid (designated dlt and dlp, respectively), appeared to be related to the thioredoxin family of thiol: disulphide oxidoreductases. The dlp gene was able to complement the DTT-sensitive phenotype, the inability to metabolize glucose 1-phosphate and the low alkaline phosphatase activity of a dsbA mutant of Escherichia coli. The dlt gene partially complemented the lack of alkaline phosphatase activity, but not the other mutant phenotypes. The products of both genes could be detected using the T7 RNA polymerase promoter expression system. The estimated molecular masses of the products of the dlt and dlp genes by SDS-PAGE were 26 and 23 kDa, respectively, the first being in agreement with the deduced amino acid sequence and the latter, somewhat smaller. The processing of a possible leader peptide in the Dlp protein, but not in the Dlt protein, could be responsible for this difference. The Dlp protein appeared as a doublet band on SDS-PAGE, which is characteristic of the oxidized and reduced states of this kind of protein.
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PMID:Homologous regions of the Salmonella enteritidis virulence plasmid and the chromosome of Salmonella typhi encode thiol: disulphide oxidoreductases belonging to the DsbA thioredoxin family. 914 3

The role of photoproduct structure, 3' --> 5' exonuclease activity, and processivity on polynucleotide synthesis past photoproducts of thymidylyl-(3' --> 5')-thymidine was investigated. Both Moloney murine leukemia virus reverse transcriptase and 3' --> 5' exonuclease-deficient (exo-) Vent polymerase were blocked by all photoproducts, whereas Taq polymerase could slowly bypass the cis-syn dimer. T7 RNA polymerase was able to bypass all the photoproducts in the order cis-syn > Dewar > (6-4) > trans-syn-II. Klenow fragment could not bypass any of the photoproducts, but an exo- mutant could bypass the cis-syn dimer to a greater extent than the others. Likewise T7 DNA polymerase, composed of the T7 gene 5 protein and Escherichia coli thioredoxin, was blocked by all the photoproducts, but the exo- mutant Sequenase 2.0 was able to bypass them all in the order cis-syn > Dewar > trans-syn-II > (6-4). No bypass occurred with an exo- gene 5 protein in the absence of the thioredoxin processivity factor. Bypass of the cis-syn and trans-syn-II products by Sequenase 2.0 was essentially non-mutagenic, whereas about 20% dTMP was inserted opposite the 5'-T of the Dewar photoproduct. A mechanism involving a transient abasic site is proposed to account for the preferential incorporation of dAMP opposite the 3'-T of the photoproducts.
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PMID:The ability of a variety of polymerases to synthesize past site-specific cis-syn, trans-syn-II, (6-4), and Dewar photoproducts of thymidylyl-(3'-->5')-thymidine. 970 33

We have identified an RNA polymerase sigma factor, sigmaR, that is part of a system that senses and responds to thiol oxidation in the Gram-positive, antibiotic-producing bacterium Streptomyces coelicolor A3(2). Deletion of the gene (sigR) encoding sigmaR caused sensitivity to the thiol-specific oxidant diamide and to the redox cycling compounds menadione and plumbagin. This correlated with reduced levels of disulfide reductase activity and an inability to induce this activity on exposure to diamide. The trxBA operon, encoding thioredoxin reductase and thioredoxin, was found to be under the direct control of sigmaR. trxBA is transcribed from two promoters, trxBp1 and trxBp2, separated by 5-6 bp. trxBp1 is transiently induced at least 50-fold in response to diamide treatment in a sigR-dependent manner. Purified sigmaR directed transcription from trxBp1 in vitro, indicating that trxBp1 is a target for sigmaR. Transcription of sigR itself initiates at two promoters, sigRp1 and sigRp2, which are separated by 173 bp. The sigRp2 transcript was undetectable in a sigR-null mutant, and purified sigmaR could direct transcription from sigRp2 in vitro, indicating that sigR is positively autoregulated. Transcription from sigRp2 was also transiently induced (70-fold) following treatment with diamide. We propose a model in which sigmaR induces expression of the thioredoxin system in response to cytoplasmic disulfide bond formation. Upon reestablishment of normal thiol levels, sigmaR activity is switched off, resulting in down-regulation of trxBA and sigR. We present evidence that the sigmaR system also functions in the actinomycete pathogen Mycobacterium tuberculosis.
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PMID:sigmaR, an RNA polymerase sigma factor that modulates expression of the thioredoxin system in response to oxidative stress in Streptomyces coelicolor A3(2). 975 77

Although NO has been postulated to play important roles in host defences, it is potentially damaging for exposed cells, including for the macrophages producing the NO. Thus a network of radical acceptors and enzymes is thought to play an important redox-buffering role to protect cells against NO-mediated injury. We examined the properties of the redox systems superoxide dismutase (SOD)/catalase, glutathione (GSH) and thioredoxin (Trx), in regulating the viability of two human monocytic cell lines (THP1 and U937) exposed to the NO-generating compound diethylene triamine-nitric oxide (DETA-NO). We observed that NO-induced cytotoxic effects were time- and dose-dependent towards the two cell lines. After vitamin-induced differentiation in vitro with retinoic acid (RA) and 1,25-dihydroxy vitamin D(3) (VD), termed RA/VD, we observed that THP1 RA/VD cells became more resistant to NO-mediated cytotoxicity whereas the susceptibility of U937 cells was not modified. Using Western blotting and reverse-transcriptase PCR methods, we observed that gene transcription and protein expression of Trx and thioredoxin reductase were significantly increased upon RA/VD treatment and differentiation in THP1 cells. By contrast, SOD/catalase and GSH redox state remained unmodified. Finally, a stable transfectant THP1 line overexpressing Trx was found to be more resistant than THP1 control cells that were untransfected or transfected with an empty plasmid, when exposed to DETA-NO in vitro. In conclusion, we observed an inverse correlation between cell susceptibility to NO damaging effects and Trx expression, suggesting that the Trx system may have important preventative capacities towards NO-mediated cellular injury in monocytic macrophage cells.
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PMID:Protective effect of thioredoxin upon NO-mediated cell injury in THP1 monocytic human cells. 1069 4

The expression of Vitreosilla hemoglobin gene (vgb) is regulated by oxygen consistence in E. coli. The gene transcription is activated under the condition of limited oxygen. A new system for expressing heterologous gene in E. coli regulated by dissolved oxygen consistence was constructed. It includes a host bacteria GJ100, which contained T7 RNA polymerase gene controlled by vgb promoter, and an expression vector on which the heterologous gene was under the control of a T7 promoter. The results indicated that E. coli thioredoxin A, IgG binding domain of Staphylococcus protein A(ZZ), snake neurotoxin, salmon calcitonin hexa-polymer, human interleukinII (IL2) and human pro-urokinase genes could be expressed efficiently. Expression level was more than 30% of the total cellular protein.
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PMID:A new system for expressing heterologous gene in Escherichia coli regulated by oxygen consistence in the environment. 1093 65

The virus-specific components (nsP1-nsP4) of Semliki Forest virus RNA polymerase are synthesized as a large polyprotein (P1234), which is cleaved by a virus-encoded protease. Based on mutagenesis studies, nsP2 has been implicated as the protease moiety of P1234. Here, we show that purified nsP2 (799 amino acids) and its C-terminal domain Pro39 (amino acids 459-799) specifically process P1234 and its cleavage intermediates. Analysis of cleavage products of in vitro synthesized P12, P23, and P34 revealed cleavages at sites 1/2, 2/3, and 3/4. The cleavage regions of P1/2, P2/3, and P3/4 were expressed as thioredoxin fusion proteins (Trx12, Trx23, and Trx34), containing approximately 20 amino acids on each side of the cleavage sites. After exposure of these purified fusion proteins to nsP2 or Pro39, the reaction products were analyzed by SDS-polyacrylamide gel electrophoresis, mass spectrometry, and amino-terminal sequencing. The expected amino termini of nsP2, nsP3, and nsP4 were detected. The cleavage at 3/4 site was most efficient, whereas cleavage at 1/2 site required 5000-fold more of Pro39, and 2/3 site was almost resistant to cleavage. The activity of Pro39 was inhibited by N-ethylmaleimide, Zn(2+), and Cu(2+), but not by EDTA, phenylmethylsulfonyl fluoride, or pepstatin, in accordance with the thiol proteinase nature of nsP2.
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PMID:Site-specific protease activity of the carboxyl-terminal domain of Semliki Forest virus replicase protein nsP2. 1141 May 98


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