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)

Poly(rA).oligo(dT)n binding to human immunodeficiency virus type-1 reverse transcriptase heterodimer (p66-p51) was primer length-dependent. The estimated Kd for (n = 10-14) was 20-30 nM and for (n = 16-20) was 0.11-0.14 nM. Gel electrophoretic analysis of the patterns of primer extension was consistent with an abrupt change in the Kd between a primer length of 14 and 16 nucleotides. Further, the rate constant for dissociation of the reverse transcriptase-template-primer complex was determined from steady state kinetics and enzyme-template-primer trapping experiments to be independent of primer length. Thus, the abrupt change in Kd was most likely due to a change in the rate constant for formation of the reverse transcriptase-template-primer complex. A similar shift in the Kd for template-primer binding was observed with poly(dA).oligo(dT)n. Reverse transcriptase homodimer (p66) catalyzed the incorporation of dTMP into poly(rA).oligo(dT)n with the same primer length dependence observed for the heterodimer. In contrast, binding of the p51 homodimer to poly(rA).oligo(dT)n was independent of primer length. Thus, the RNase H domain may contribute to reverse transcriptase heterodimer or p66 homodimer binding to template-primers in which the primer length is greater than 14 nucleotides.
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PMID:Human immunodeficiency virus reverse transcriptase. Effect of primer length on template-primer binding. 171 16

The rfbB gene (dThymidine-diphospho-D-glucose-4,6-dehydratase) from Salmonella serovar typhimurium LT2 was cloned and over-expressed using the T7 RNA polymerase/promoter system. The expressed protein, which represents almost 10% of the total cellular protein was purified 14-fold. dTDP-D-glucose 4,6-dehydratase is a homodimer of 43 kDa subunits, is highly specific for dTDP-D-glucose and shows a Km of 427 microM and Vmax of 0.93 mu moles min-1 micrograms-1 of protein for dTDP-D-glucose. The N-terminal analysis confirmed the start position of the gene in the DNA sequence. Complete deactivation of the enzyme by the addition of p-chloromercurisulfonic acid and total reactivation by the addition of mercaptoethanol, co-factor NAD+ and cystein showed that a -SH group of the cysteine is involved in the catalytic site.
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PMID:High level expression and purification of dthymidine diphospho-D-glucose 4,6-dehydratase (rfbB) from Salmonella serovar typhimurium LT2. 199 76

DNA-dependent RNA polymerase from Escherichia coli contains 2 mol of zinc/mol of holoenzyme (alpha 2 beta beta' sigma) with one zinc each in the beta and beta' subunits. A new method to substitute selectively the zinc in the beta subunit was developed by the inactivation of RNA polymerase with 0.25 M NaNO3, 1 M NaCl, 1 mM diaminocyclohexane tetraacetic acid, and 0.1 mM dithiothreitol followed by reconstitution with Co(II), Cd(II), or Cu(II). The hybrid Co-Zn, Cd-Zn, or Cu-Zn RNA polymerase thus obtained retains, respectively, 91, 88, and 50% enzyme activity of the reconstituted Zn-Zn RNA polymerase. Co-Zn RNA polymerase exhibits absorption maxima at 395 and 465 nm, and Cu-Zn RNA polymerase at 637 nm (epsilon = 815 M-1 cm-1). 1-Aminonaphthalene-5-sulfonic acid (AmNS) derivatives of ATP, UTP, and dinucleoside monophosphates (diNMPs), UpA or ApU, were synthesized with AmNS attached to NTP via a gamma-phosphoamidate bond or to diNMPs via a 5'-secondary amine linkage. Since the fluorescence emission maxima of (5'-AmNS)UpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP at 445, 464, and 464 nm, respectively, when excited at 340 nm, overlap the 465-nm absorption band of Co-Zn RNA polymerase, the spatial relationship between fluorescence substrate analogs and the intrinsic Co(II) in Co-Zn RNA polymerase was studied by fluorescence resonance energy transfer technique. The fluorescence of the initiator, (5'-AmNS)UpA, and elongator, (gamma-AmNS)UTP, of the RNA chain, was quenched 20.3 and 7.1%, by the addition of saturation concentration of Zn-Zn RNA polymerase, and 21.3 and 14.7%, respectively, by the addition of template, poly(dA-dT). The fluorescence of (5'-AmNS)UpA and (gamma-AmNS)UTP was quenched 81.8 and 80.6%, respectively, by the addition of the saturation concentration of Co-Zn RNA polymerase in the absence of template, and 82.7 and 82.9% in the presence of template. On the basis of respective Ro values of 21.3 and 21.9 A for the (5'-AmNS)UpA-Co and (gamma-AmNS)UTP-Co pairs, the distances from Co(II) to the initiation site and to the elongation site were calculated to be 17.4 and 17.5 A, respectively, in the absence and 17.2 and 17.4 A in the presence of template.
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PMID:Fluorescence resonance energy transfer studies on the proximity relationship between the intrinsic metal ion and substrate binding sites of Escherichia coli RNA polymerase. 330 70

More than 40 protein species including RNA polymerase were found to be phosphorylated in Escherichia coli on analyses of 32P-labeled cell lysates by single and two-dimensional gel electrophoresis and autoradiography. The protein species and the level of phosphorylation varied depending on the cell growth phase. With [gamma-32P]ATP as a substrate, cell lysates phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. Both serine and threonine were the major phosphate acceptors in whole cell lysates. Starting from a partially purified RNA polymerase preparation with the protein phosphorylation activity and using an E. coli protein with an apparent Mr = 90K (K represents X 1000) as the substrate, we purified a protein kinase with a native Mr approximately 120K to apparent homogeneity. The protein kinase is either a heterodimer of 61K and 66K polypeptides or a homodimer of one of these polypeptides. We also isolated a 100K protein with self-phosphorylation activity.
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PMID:Protein phosphorylation in Escherichia coli and purification of a protein kinase. 636 41

A simple in vitro substitution method involving a sequential denaturation--reconstitution process was developed to substitute selectively one of the two intrinsic Zn ions in Escherichia coli RNA polymerase with Co, Mn, Ni, or Cu ion. The resultant metal hybrid Co-Zn, Mn-Zn, Ni-Zn, and Cu-Zn RNA polymerases possess 100, 100, 60, and 17% of the enzymatic activity of the reconstituted Zn-Zn enzyme, respectively. The substituted metal was found to be located in the beta subunit of the polymerase which contains the substrate binding site. The biochemical and physical properties of these metal-substituted polymerases were compared with those of the native Zn enzyme. Co-Zn and Ni-Zn core polymerases exhibit characteristic absorption spectra in the near-UV and visible region, while Mn-Zn and Cu-Zn enzymes do not. The Co-Zn enzyme shows two major peaks at 400 nm (epsilon = 3000) and 475 nm (epsilon = 2700), while the Ni-Zn enzyme exhibits a major peak at 462 nm (epsilon = 8000). The difference absorption spectrum of Ni-Zn core polymerase could be perturbed by the addition of substrate ATP but not by UTP in the absence of template and Mg(II) ion. These observations suggest that the substituted metal was located at the initiation site of the enzyme. The various metal hybrid enzymes do not differ appreciably in their abilities to incorporate noncomplementary nucleotide or deoxyribonucleotide into RNA product. It was found, however, that the difference in enzymatic activities of these metal hybrid enzymes resides at least partly in the initiation step of RNA synthesis.
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PMID:Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals. 675 22

SoxR protein of Escherichia coli is activated by superoxide-generating agents or nitric oxide as a powerful transcription activator of the soxS gene, whose product activates approximately 10 other promoters. SoxR contains non-heme iron essential for abortive initiation of transcription in vitro. Here we show that this metal dependence extends to full-length transcription in vitro. In the presence of E. coli sigma 70 RNA polymerase, iron-containing SoxR mediates open complex formation at the soxS promoter, as determined using footprinting with Cu-5-phenyl-1,10-phenanthroline. We investigated the nature of the SoxR iron center by chemical analyses and electron paramagnetic resonance spectroscopy. Dithionite-reduced Fe-SoxR exhibited an almost axial paramagnetic signature with g values of 2.01 and 1.93 observable up to 100 K. These features, together with quantitation of spin, iron, and S2-, and hydrodynamic evidence that SoxR is a homodimer in solution, indicate that (SoxR)2 contains two [2Fe-2S] clusters. Treatment of Fe-SoxR with high concentrations of dithiothreitol caused subtle changes in the visible absorption spectrum and blocked transcriptional activity without generating reduced [2Fe-2S] centers, but was also associated with the loss of iron from the protein. However, lowering the thiol concentration by dilution allowed spontaneous regeneration of active Fe-SoxR.
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PMID:Binuclear [2Fe-2S] clusters in the Escherichia coli SoxR protein and role of the metal centers in transcription. 767 13

SoxR protein governs the soxRS (superoxide response) regulon of Escherichia coli by becoming a transcriptional activator when the cells are exposed to compounds that mediate univalent redox reactions, many of which produce superoxide as a by-product. SoxR was overproduced and purified to near homogeneity from a strain bearing an expression vector. It could bind specifically to the soxS operator even in the absence of RNA polymerase. The aerobically purified protein, which is readily autooxidized, could activate the transcription of soxS DNA even without exposure to known inducing agents. SoxR is a globular homodimer. It contains one [2Fe-2S] cluster per polypeptide chain, as demonstrated by optical and EPR spectroscopy combined with stoichiometric analysis of iron content, unpaired-electron-spin density, and reduction by dithionite. The protein is active in its oxidized ([2Fe-2S]2+) state. The presence of a prosthetic group capable of univalent redox reactions may help to explain the activation of the regulon in vivo by compounds that can mediate such reactions.
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PMID:Overproduction and physical characterization of SoxR, a [2Fe-2S] protein that governs an oxidative response regulon in Escherichia coli. 773 Mar 38

This paper describes the cytotoxicity of ranunculin (RAN) and its mechanism of action. The IC50 of RAN against the KB and Bel7402 cells in colony test were found to be 0.21 and 0.35 mumol/L respectively. RAN inhibited the incorporation of 3H-labeled precursors into DNA and RNA of L1210 cells. RAN (15 mumol/L) markedly decreased DNA synthesis catalyzed by DNA polymerase I and promoted the generation of superoxide anions in DMSO/KO2 system. In the meantime, SOD and CAT were shown to partly revoke the inhibitory effects of RAN upon the incorporation of 3H-TdR into DNA. No direct reaction between RAN and DNA template was observed and no effect of RAN on DNA TOPO II or RNA polymerase was found. Our results suggest that the cytotoxicity of RAN in vitro may be due to inhibition of DNA polymerase and increase of oxygen free radicals.
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PMID:[The cytotoxicity and action mechanism of ranunculin in vitro]. 823 75

The TyrR protein of Escherichia coli K12 is a homodimer containing 513 amino acids/subunit. This protein is important in the transcriptional regulation of several genes whose protein products catalyze steps in aromatic amino acid biosynthesis or transport. Methods were developed for efficiently purifying the TyrR protein to apparent homogeneity. We analyzed the pattern of cleavage of the TyrR protein by trypsin, either in the absence of ligands or in the presence of saturating levels of L-tyrosine, ATP, or poly(dI-dC). At low (1:200 ratio by weight) trypsin levels, in the absence of ligands, two major digestion products accumulated. These were polypeptides of 22 and 31 kDa, shown to contain amino acid residues 1-190 and 191-467, respectively. The pattern of trypsin cleavage was unaffected by tyrosine. In the presence of ATP, an intermediate species of 53 kDa, probably containing amino acid residues 1-467, was observed. The kinetics of appearance of the 53-kDa species were consistent with a role for ATP in accelerating the hydrolysis of the R467-F468 peptide bond. The 53-kDa polypeptide underwent further tryptic hydrolysis to yield fragments of 22 and 31 kDa. When both tyrosine and ATP were present, the rate of formation of the 22- and 31-kDa fragments was more rapid than in the absence of these ligands. It appears that when both ligands are bound, the rates of hydrolysis of peptide bonds R190-Q191 and R467-F468 are both enhanced. Additional limited proteolysis experiments suggested that polypeptide segment 191-467 contains ATP binding site(s), and that the rate of cleavage of peptide bonds R190-Q191 and R467-F468 is altered when the TyrR protein interacts with poly(dI-dC), an analog of target DNA. Our results reveal the presence of two major structural domains within the TyrR protein. The first domain (amino acid residues 1-190) is extremely resistant to hydrolysis by trypsin. The second domain (residues 191-467), which is likely to contain ATP-binding site(s), is homologous to several other transcriptional activators specific for promoters responsive to the sigma 54 form of RNA polymerase. The remainder of the TyrR protein (residues 468-513) contains the operator recognition elements, probably arranged in the form of a helix-turn-helix motif. This polypeptide segment was not detected as a discrete tryptic hydrolysis product.
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PMID:The TyrR protein of Escherichia coli, analysis by limited proteolysis of domain structure and ligand-mediated conformational changes. 844 80

The IL-1R antagonist protein (IRAP) is a competitive inhibitor of IL-1, which is predominantly synthesized by monocytes. We show that this molecule is also expressed in human synovial fibroblasts and dermal fibroblasts (CRL 1445). IRAP mRNA was regulated in a time- and dose-dependent manner by IL-1 alpha, TNF-alpha, LPS, and PMA. Maximal induction of IRAP mRNA was observed between 8 and 16 h after stimulation with IL-1 alpha (1 U/ml), TNF-alpha (10 U/ml), LPS (50 ng/ml), and PMA (10 ng/ml). Their relative efficacy was as follows: PMA > LPS > IL-1 alpha > TNF-alpha. Potentiation was observed when fibroblasts were treated with IL-1 alpha plus basic fibroblast growth factor and IL-1 alpha plus platelet-derived growth factor-BB homodimer. Although LPS and PMA were the best inducers of IRAP mRNA, quantitation of the IRAP protein revealed that its synthesis and release were differentially regulated. Immunoprecipitation and SDS-PAGE of culture supernatant from LPS-treated cells and cell lysates of fibroblasts treated with LPS or PMA showed a single IRAP band with a molecular mass of approximately 22 kDa. Very little IRAP was detected in culture supernatants of cells treated with PMA. Quantitation of IRAP revealed that LPS induced the synthesis of secreted IRAP that was released, whereas the majority of the protein induced by PMA remained cell-associated. Reverse transcriptase-polymerase chain reaction amplification demonstrated that although LPS and PMA induced both transcripts, LPS preferentially induced secreted IRAP, whereas PMA differentially induced intracellular IRAP mRNA. Fibroblasts synthesize at least two different forms of IRAP depending on the inducing signal, and may regulate the inflammatory response by dampening the proinflammatory effects of IL-1 via a negative feedback mechanism with IRAP. The relative importance of fibroblast sIRAP vs intracellular IRAP in regulating the inflammatory response by the connective tissue remains to be determined.
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PMID:Regulation of expression of IL-1 receptor antagonist protein in human synovial and dermal fibroblasts. 847 46


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