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)

Partially purified DNA-dependent RNA polymerase of Streptomyces granaticolor was further separated on phosphocellulose in 50% glycerol and a single activity peak was obtained. The enzyme isolated in this way consisted of 4 main proteins with molar mass of 145, 132, 50 and 46 kg/mol. These four subunits represented 93% proteins of the active fraction. To test the ability of RNA polymerase to recognize specific sites on DNA, binding sites for RNA polymerase on phage phi 29 DNA were mapped by electron microscopy. The specific binding sites detected were compared with those for RNA polymerases from Escherichia coli and Bacillus subtilis.
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PMID:Isolation of DNA-dependent RNA polymerase from Streptomyces granaticolor and its binding to phage phi 29 DNA. 182 45

The promoter region preceding the hutUH operon in Klebsiella aerogenes contains two oppositely oriented, overlapping promoters. In the absence of catabolite gene activator protein-cyclic AMP (CAP-cAMP), transcription proceeds primarily from the backward-oriented promoter (Pc), whose function has not yet been determined, and only very weakly from the forward hutUH promoter, hutUp. In the presence of CAP-cAMP, Pc is repressed and transcription from hutUp is favored. Two protein components required for this in vitro transcription system, RNA polymerase (RNAP) and CAP, were purified from K. aerogenes and were shown to be functionally interchangeable with the corresponding proteins from Escherichia coli, suggesting that E. coli RNAP could be used to study some aspects of hut transcription. We showed that a gradual activation of hutUp (by increasing concentrations of CAP, cAMP, or glycerol) resulted in a parallel repression of Pc, arguing in favor of a direct competition between the two promoters. The presence of a DNA sequence resembling the consensus for CAP-binding sites and centered at nucleotide -82 (relative to hutUp) initially suggested that a primary role of CAP was to repress Pc, thereby indirectly activating hutUp. However, the relatively slow formation of open complexes at Pc, even in the absence of CAP-cAMP, showed that Pc is a weak promoter and likely to be a poor competitor for RNAP. The observed dominance of Pc over hutUp suggested that the latter is an even weaker promoter. Thus, repression of Pc would not be sufficient to cause the observed increase in hutUp activity, and the CAP-cAMP complex must play a direct role in the activation of hutUp.
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PMID:In vitro transcription of the histidine utilization (hutUH) operon from Klebsiella aerogenes. 184 33

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19 + 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115-120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19 + 2.1. Primers synthesized using M13mp19 + 2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.
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PMID:Pea chloroplast DNA primase: characterization and role in initiation of replication. 186 57

Heat treatment of yeast nuclear extracts abolished the capacity to initiate transcription at RNA polymerase II promoters. Activity was restored by the addition of both recombinant yeast TFIID and partially purified factor b, a yeast fraction shown previously to be required for polymerase II transcription. On the basis of this assay with heat-treated extract, factor b was purified to virtual homogeneity. The factor appears to comprise polypeptides of approximately 85, 75, and 50 kDa, since these three polypeptides co-purify with activity, and since a native mass of about 200 kDa is estimated from glycerol gradient sedimentation and gel filtration.
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PMID:Purification and characterization of yeast RNA polymerase II transcription factor b. 191 15

The genes for the peripheral glycerol carbon metabolic pathway (glp) in Pseudomonas aeruginosa are postulated to be positively regulated by GlpR. A gene complementing the glpR2 allele, affecting expression of the putative activator, was cloned by a bacteriophage mini-D3112-based in vivo cloning method. Mini-D3112 replicons were isolated by transfecting glpR2 strain PRP406 and selecting clones able to grow on minimal medium containing glycerol as the sole carbon and energy source. Preliminary biochemical characterization indicated that the cloned activator gene for glycerol metabolism (agmR) may not be allelic to glpR. Restriction analysis and recloning of DNA fragments located the agmR gene to a 2.3-kb EcoRV-SstI DNA fragment. In a T7 RNA polymerase expression system, a single 26,000-Da protein was expressed from this DNA fragment. The amino acid sequence of this protein, deduced from the nucleotide sequence reported here, demonstrates its homology to the effector (or regulator) proteins of the environmentally responsive two-component regulators. The carboxy-terminal region of AgmR contains a possible helix-turn-helix DNA-binding motif and resembles sequences found in transcriptional regulators of the LuxR family.
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PMID:The agmR gene, an environmentally responsive gene, complements defective glpR, which encodes the putative activator for glycerol metabolism in Pseudomonas aeruginosa. 193 86

The iclR gene of Escherichia coli K-12, which encodes a regulatory protein (repressor) for the aceBAK operon, is located between that operon and metH in the 91-min region of the chromosome. The iclR gene was cloned and expressed in a coupled T7 RNA polymerase/promoter system and the gene product was identified by specific binding to a fragment containing the aceBAK operator region. The iclR gene product is a polypeptide of 274 amino acids (aa) with a calculated Mr of 29,741. Comparison of the deduced IclR aa sequence to that of Salmonella typhimurium revealed that the two IclR repressors exhibit 89% identity. A possible helix-turn-helix motif characteristic of DNA-binding proteins was found within the IclR sequence. A search in protein data banks revealed that IclR has a score of similarity of 43.7% with GylR, a transcriptional regulator of the glycerol operon of Streptomyces coelicolor.
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PMID:Overproduction and characterization of the iclR gene product of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2. 199 31

Human interleukin-6 or B-cell stimulatory factor-2 is a cytokine involved in acute phase and immune response. Cloning of cDNA for human interleukin-6 in the pT7.7 expression plasmid under the control of a bacteriophage T7 RNA polymerase promoter system allows rapid production of the cytokine in Escherichia coli. Upon cell induction with isopropyl thiogalactopyranoside, recombinant human interleukin-6 is overexpressed and forms insoluble inclusion bodies. Solubilization of the protein with 6 M guanidine hydrochloride and refolding in the presence of a reduction/oxidation system results in a quantitative recovery of recombinant human interleukin-6. This material is already 70% pure and can be further purified to homogeneity with a single passage over a weak anionic-exchange column. Extended structural characterization of the purified protein by electrospray mass spectrometry, automated Edman degradation and peptide mapping by high-pressure liquid chromatography/fast-atom-bombardment mass spectrometry demonstrates that recombinant human interleukin-6 is identical to the natural protein both in amino acid sequence and S-S bridge content. However, it contains a minor component accounting for about 20% of the entire translated protein which exhibits a Met-Ala dipeptide extension at the N-terminus. Purified recombinant human interleukin-6 is biologically active because it is able to induce at least 70-fold the human C-reactive promoter transfected in human hepatoma Hep 3B cells and is stable for several months in 10% glycerol at 4 degrees C. The expression system described in the present work has the main advantage of producing a high yield of recombinant human interleukin-6 (about 25 mg/l) combined with a very simple purification scheme.
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PMID:Single-step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector. 205 Jan 35

The polypeptide encoded by the vaccinia virus open reading frame D7R was synthesized in bacteria. Immunization of rabbits with the polypeptide resulted in antibodies that specifically recognized a virion polypeptide of 20,000 daltons. The immunoreactivity with the 20,000-dalton polypeptide was found to coincide with the virion-associated DNA-dependent RNA polymerase through DEAE-cellulose chromatography and glycerol gradient sedimentation. These results argue that the product of the vaccinia open reading frame D7R is a subunit of the viral RNA polymerase.
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PMID:Vaccinia virus gene D7R encodes a 20,000-dalton subunit of the viral DNA-dependent RNA polymerase. 221 12

Various agents were tested for their effects on microbial proteases, which activity was monitored by the analysis of cleaved peptide bands in SDS-polyacrylamide gel electrophoresis. Using casein as a substrate, fungal protease (type XIX) was inhibited by the phenyl methyl sulphonyl fluoride, chymostatin, antipain and leupeptin, while bacterial protease (type XXVI) was inhibited by phosphatidyl glycerol, phosphatidyl inositol and sphingosine. MS2 RNA exerted minor inhibition on the bacterial proteolysis of regulatory subunits of cyclic AMP-dependent protein kinase (A-PK). The cleavage of DNA binding protein by both proteases was inhibited, in the presence of MS2 RNA and lambda DNA. In comparison, phosphatidyl serine slightly stimulated the fungal protease on the cleavage of ribonuclease T1. RNA polymerase is a good substrate of the bacterial protease as indicated by the generation of multiple cleaved peptide fragments, whereas alkaline phosphatase is not susceptible to proteolysis.
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PMID:A further study on the regulation of microbial proteases. 222 36

The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter. The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide approximately 70 nM). Glycerol gradient analysis of the E. coli lystate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins. However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lystate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2nM). Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor. Radiosequence analysis of the recombinant steroid-binding domain expressed in E. coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly. However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.
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PMID:The steroid-binding properties of recombinant glucocorticoid receptor: a putative role for heat shock protein hsp90. 227 31


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