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)

DNA-dependent RNA polymerase was solubilized from nuclei of Ehrlich ascites carcinoma (EAC) cells by sonic disruption in the presence of 0.3 M (NH4)2 SO4, and the multiple RNA polymerases were separated by chromatography on DEAE-Sephadex A-25. Elution with a nine-step gradient of (NH4)2 SO4 yielded five peaks of activity designated RNA polymerases Ia, Ib, IIa, IIb, and III, of which IIb was the most prominent. Linear-gradient elusion also yielded five peaks of the same designation, but Ia and Ib, as well as IIa and IIb, were not well separated. IIa and IIb were inhibited completely by 0.1 mug alpha-amanitin/ml, whereas the other forms were not. EAC RNA polymerases Ia, Ib, IIa, and IIb possessed Mg2+ ion, Mn2+ ion, and (NH4)2 SO4 optima, molecular weights, and thermal sensitivities similar to those reported for other mammalian DNA-dependent RNA polymerases. As measured by relative ribonucleoside monophosphate incorporation, with native calf thymus DNA template, EAC RNA polymerases Ia and Ib synthesized ribosomal RNA-like products, whereas forms IIa, IIb, and the parent enzyme mixture synthesized compounds that were more similar to DNA. No species specificity was found for DNA templates, and denatured DNA was consistently preferred to the native template by RNA polymerases IIa and IIb; the two kinds of template were about equally efficient for RNA polymerases Ia and Ib. Although EAC RNA polymerases Ia, IIa, and IIb were inhibited by daunomycin, form IIa was preferentially affected. 3',5'-Cyclic AMP, 3',5'-Cyclic GMP, and gibberellic acid, implicated as RNA polymerase regulators in other systems, were generally ineffective. The levels of nuclear RNA polymerase activities, per mg DNA, of 3 mouse ascites tumors (EAC, 6C3HED lymphosarcoma, and TA3 adenocarcinoma) were compared with those from 3 normal mouse tissues (kidney, liver, and spleen). On the average, the tumor cell nuclei contained (per mg of DNA) 8.9, 1.5, 2.7, 20.0, and 3.8 times the activities of RNA polymerases Ia, Ib, IIa, IIb, and III, respectively, as the normal cells, but the difference was significantly only for IIb.
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PMID:DNA-dependent RNA polymerases of Ehrlich carcinoma, other murine ascites tumors, and murine normal tissues. 117 42

The DNA-dependent syntheses of different enzymes of the bacteriophages T3 and T7 were studied in an Escherichia coli system in vitro with respect to the optimal Mg2+ concentration and its interdependence with substituting (e.g. spermidine) and complexing agents (e.g. phosphoenolpyruvate). The following results were obtained. 1. The optimal conditions for the syntheses of the different enzymes were not identical. The optima for RNA polymerase synthesis were 8 mM Mg2+, 10 mM P-pyruvate and 3 mM spermidine; for S-adenosyl-L-methionine cleaving enzyme synthesis, 6 mM Mg2+, 6 mM P-pyruvate and 3 mM spermidine; and for lysozyme synthesis, 13-18 mM Mg2+, 28 mM P-pyruvate and 3-0 mM spermidine. 2. The optimal conditions for the synthesis of analog enzymes (RNA polymerases and lysozymes) from the two templates were identical with experimental error. 3. Mg2+ and spermidine substituted for each other in relation to the number of their charges. 4. The apparent complexing of one Mg2+ molecule required the addition of 3-5 P pyruvate molecules. 5. Under the optimal conditions the enzyme-synthesizing activity was higher by more than a factor of 10 compared to previously described systems.
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PMID:The interdependence of magnesium with spermidine and phosphoenolpyruvate in an enzyme-synthesizing system in vitro. 126 43

The in vivo observation that the expression of bacteriophage T7 gene 3.5 (T7 lysozyme) inactivates T7 class II transcription and the in vitro observation that T7 lysozyme inhibits T7 RNA polymerase lead to the hypothesis that T7 lysozyme might preferentially inhibit transcription from T7 class II promoters. T7 lysozyme was cloned into a lambda pL expression vector, overproduced in Escherichia coli, and purified. The ability of purified T7 lysozyme to inhibit transcription from T7 DNA, the cloned T7 class II promoters, phi 2.5 and phi 4.7, and the cloned class III promoter, phi 10, was measured in vitro. It was observed that the effectiveness of T7 lysozyme as an inhibitor of T7 RNA polymerase is inversely related to the concentration of Mg2+; T7 lysozyme inhibits T7 RNA polymerase most effectively at low Mg2+ concentrations. In addition, no preferential inhibition of transcription from cloned T7 class II promoters was observed, nor was a strong T7 class III promoter preferred when transcriptional capacity was reduced by T7 lysozyme. These observations contradict the hypotheses that the temporal control of T7 gene expression is either due to direct and selective inhibition of the T7 class II promoters by T7 lysozyme or to preferential transcription of the strong T7 class III promoters when transcriptional capacity is reduced by T7 lysozyme. It appears that alternative mechanisms such as the involvement of additional proteins and/or cellular conditions to enhance transcription from T7 class III promoters or to inhibit transcription from T7 class II promoter are necessary to explain the temporal control of transcription of bacteriophage T7.
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PMID:Inhibition of T7 RNA polymerase by T7 lysozyme in vitro. 135 74

DNA-dependent RNA polymerase (EC 2.7.7.6) was purified from Pseudomonas putida. The enzyme had the typical composition of beta',beta,alpha, and sigma subunits of eubacterial RNA polymerases. The molecular masses of the subunits were 156,000 Da, 151,000 Da, 87,000 Da, and 42,000 Da, respectively, as measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The NH2-terminal amino acid residues of the alpha subunit had a marked homology with those of the alpha subunit of Escherichia coli RNA polymerase. The enzyme activity was dependent on ribonucleoside triphosphates, Mg2+, and a DNA template, and was inhibited in vitro by rifampicin. The enzyme activity was maximal in the presence of 10 mM MgCl2. In an in vitro transcription assay using the tac promoter-controlled DNA as a template, the RNA polymerase of P. putida initiated transcription at the same site as that of E. coli.
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PMID:Purification and characterization of a DNA-dependent RNA polymerase from Pseudomonas putida. 136 75

Escherichia coli RNA polymerase can terminate transcription efficiently at rho-independent terminators in a purified transcription system in the absence of accessory factors. This process of "intrinsic termination" involves direct recognition of the terminator by the core RNA polymerase, and provides an important model system for the study of the molecular interactions involved in the switch between elongation and termination. We have analyzed the intrinsic termination efficiency (%T) of 13 rho-independent terminators, under a variety of in vitro reaction conditions. Although all of these sites share the general sequence features of typical rho-independent terminators, we find a wide range of %T (2% to 90%) for the different sites under our standard transcription conditions. While %T for a particular site is characteristic of that site, the efficiency can be altered considerably by the nature and concentration of salts in the reaction, by alteration of the concentrations of the nucleoside triphosphate substrates, or by transcription from supercoiled rather than linear templates. Surprisingly, different conditions can alter %T to a different extent for different terminators. For neutral salts such as potassium chloride or potassium glutamate, changes in the range from 0.1 to 1 M affect %T for different terminators in a distinct manner, depending on the terminator and the anion involved. At some sites, %T is greatly increased by Cl- concentrations up to 1 M, while at other sites %T is reduced or unaffected by these conditions. At some sites K+ concentrations up to 1 M give a modest increase in %T, while at other sites %T is slightly reduced under the same conditions. Thus the actual values of %T, as well as the order of terminator sites ranked according to %T, can be altered greatly according to the choice of reaction conditions. Reduction of the Mg2+ concentration below 1 mM has a dramatic and quite different effect, enhancing termination to approximately 100% for all terminators tested. Transcription of supercoiled DNA templates gives somewhat reduced %T as compared with linear DNA templates. However, the effect is no greater than twofold. Our results are not consistent with those expected for models in which %T is determined by the differential stability of DNA, RNA and hybrid duplex structures at the melted region in the transcription complex. Thus, the Cl anion does not affect the stability of nucleic acid duplexes even at 1 M concentrations, but can enhance termination tenfold. Also, the alterations of monovalent cation concentration that affect %T are not expected to have a differential effect on Tm for DNA, RNA and hybrid duplexes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators. 137 65

Wheat-germ RNA polymerase II is able to catalyse a DNA-dependent reaction of RNA synthesis in the presence of a high concentration (1 mg/ml) of the fungal toxin alpha-amanitin. This anomalous reaction is specifically directed by single-stranded or double-stranded homopolymer templates, such as poly(dC) or poly(dC).poly(dG), and occurs in the presence of either Mn2+ or Mg2+ as the bivalent metal cofactor. In contrast, the transcription of other synthetic templates, such as poly(dT), poly(dA).poly(dT) or poly[d(A-T)] is completely abolished in the presence of 1 microgram of alpha-amanitin/ml, in agreement with well-established biochemical properties of class II RNA polymerases. Size analysis of reaction products resulting from transcription of (dC)n templates of defined lengths suggests that polymerization of RNA chains proceeds through a slippage mechanism. The fact that alpha-amanitin does not impede this synthetic reaction implies that the amatoxin interferes with the translocation of wheat-germ RNA polymerase II along the DNA template.
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PMID:A DNA-dependent RNA synthesis by wheat-germ RNA polymerase II insensitive to the fungal toxin alpha-amanitin. 137 42

Comparative studies of the effects of Mg2+ vs Na+ and of acetate (OAc-) vs Cl- on the kinetics of formation and dissociation of E. coli RNA polymerase (E sigma 70)-lambda PR promoter open complexes have been used to probe the mechanism of this interaction. Composite second-order association rate constants ka and first-order dissociation rate constants kd, and their power dependences on salt concentration SKa (SKa identical to d log ka/d log [salt]) and Skd (Skd identical to d log kd/d log [salt]), were determined in MgCl2 and NaOAc to compare with the results of Roe and Record (1985) in NaCl. Replacement of NaCl by MgCl2 reduces the magnitude of Ska 2-fold (Ska = -11.9 +/- 1.1 in NaCl; Ska = -5.2 +/- 0.3 in MgCl2) and (by extrapolation) drastically reduces the magnitude of ka at any specified salt concentration (e.g., approximately 10(6)-fold at 0.2 M). Replacement of NaCl by NaOAc does not significantly affect Ska (Ska = -12.0 +/- 0.7 in NaOAc) and (by extrapolation) increased ka by approximately 80-fold at any fixed [Na+]. In the absence of Mg2+, replacement of NaCl by NaOAc is found to increase the half-life of the open complex by approximately 560-fold at fixed [Na+] without affecting Skd [Skd = 7.6 +/- 0.1 in NaOAc; in NaCl, Skd = 7.7 +/- 0.2 (Roe & Record, 1985)]. Replacement of NaCl by MgCl2 drastically reduces both Skd and the half-life of the open complex at any salt concentration below approximately 0.2 M. Strikingly, Skd = 0.4 +/- 0.1 in MgCl2, indicating that the net uptake of Mg2+ ions in the kinetically significant steps in dissociation of the open complex is much smaller than that expected by analogy with the uptake of approximately 8 Na+ ions in the corresponding steps in NaCl. In NaCl/MgCl2 mixtures, at a constant [NaCl] in the range 0.1-0.2 M, initial addition of MgCl2 (0.5 mM less than or equal to [MgCl2] less than or equal to 1 mM) increases the half-life of the open complex; further addition of MgCl2 causes the half-life to decrease, though the effect of [MgCl2] on kd is always less than that predicted by a simple competitive model. The observed effects of MgCl2 on Skd and kd differ profoundly from those expected from the behavior of kd and Skd in NaCl and NaOAc and indicate that the role of Mg2+ in dissociation is not merely that of a nonspecific divalent competitor with RNAP for interactions with DNA phosphates and of a DNA helix-stabilizer, both of which should cause kd to increase monotonically with increasing [Mg2+].(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Roles of Mg2+ in the mechanism of formation and dissociation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter: kinetic evidence for a second open complex requiring Mg2+. 138 21

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is a bifunctional enzyme synthesized as a 37-kDa precursor that is imported into the mitochondria of embryonic and transformed mammalian cells. The cDNA encoding the human bifunctional enzyme was modified to remove nucleotides corresponding to the mitochondrial targeting sequence and was subcloned into a procaryotic expression vector under the control of the T7 RNA polymerase promoter. The soluble dehydrogenase-cyclohydrolase was expressed in Escherichia coli at levels up to 150-fold higher than those found in transformed mammalian cells. Forms of the recombinant enzyme with one, three, or seven additional amino-terminal residues were purified to homogeneity and shown to have similar kinetic properties. Investigation of the absolute requirement of the enzyme for Mg2+ using fluorescence quenching indicates that this ion binds in the absence of substrates.
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PMID:Expression of human NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in Escherichia coli: purification and partial characterization. 139 22

The general transcription factor TFIIIC is necessary for transcription initiation by RNA polymerase III. TFIIIC binds predominantly to the B-Block promoter element, which is present in tRNA genes, several viral RNA genes and repetitive DNA elements, and to the TFIIIA.DNA complex on 5 S RNA genes. Here we report a characterization of Xenopus laevis TFIIIC and its interaction with the TFIIIA.5 S RNA gene complex. A polypeptide with apparent molecular mass of 85 kDa was specifically cross-linked to a B-Block oligonucleotide by UV light. This polypeptide was present in the partially purified TFIIIC fraction and in a complex with a B-Block double-stranded oligonucleotide isolated by nondenaturing gel electrophoresis. TFIIIC.TFIIIA.DNA gel mobility shift complexes were obtained using B-Block DNA affinity-purified TFIIIC and buffer conditions employing low Mg2+ (1 mM) and high dithiothreitol (7 mM) concentrations. Three TFIIIC.TFIIIA.5 S RNA gene complexes were observed by gel mobility shift analysis. One of these complexes was resistant to dissociation by the addition of competing DNA, but the formation of all three complexes was prevented by the inclusion of excess specific competitor DNA in the initial binding reactions. The apparent affinity of TFIIIC for the TFIIIA.5 S DNA complex was 5-fold higher for the somatic-type 5 S RNA gene than for the oocyte-type 5 S RNA gene. Mutations near the 5' boundary of the TFIIIA binding site alter the DNase I footprint of the TFIIIA.DNA complex and reduce the affinity of TFIIIA-mutant 5 S gene complexes for TFIIIC. Differences in TFIIIC affinity for the two classes of 5 S RNA genes may play a role in the developmental regulation of these gene families.
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PMID:Interaction of Xenopus TFIIIC with the TFIIIA.5 S RNA gene complex. 151 47

The possible regulatory interactions of purified ornithine decarboxylase with DNA-directed RNA polymerases in isolated macronuclei from the ciliated protozoan Tetrahymena thermophila were studied. It has been found that highly purified ODC (specific activity 10.2 mumols CO2 x h-1 x mg-1), even at activities of 37,500 nmol CO2 x h-1 per ml failed to alter RNA polymerase activity in the in vitro transcription assay in the presence or absence of the substrate L-ornithine at 20mM. The naturally occurring di- and polyamines putrescine, spermidine, and spermine stimulated in-vitro-transcription in isolated macronuclei more at optimal Mg2+/Mn(2+)-concentrations than at suboptimal concentrations, suggesting that polyamines act via a mechanism which is distinct from that of the inorganic cations. Of the monovalent amine compounds tested, (NH4)+ at high concentrations between 40 and 50mM slightly stimulated activity whereas the onset of stimulation by the organic amine compounds, piperidine and cyclohexylamine, was inversely related to the hydrophobicity of each particular compound. In the series of divalent amines, the correct distance between the N-atoms appeared to be very important since ethylenediamine and piperazine did not stimulate significantly but did inhibit at concentrations above 5 mM. 1,3-Diaminopropane stimulated slightly but inhibited above 10 mM, whereas the 1,4-diamino compounds putrescine and 1,4-diaminocyclohexane (DAC) were equally potent stimulators with the more hydrophobic one, DAC, reaching the maximum at lower concentrations than putrescine. For the trivalent amines, the influence of correct spacing seems not to be as important: N-(2-aminoethyl)piperazine stimulated very similar to spermidine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of DNA-directed RNA polymerases in isolated macronuclei of the ciliated protozoan Tetrahymena thermophila. Effects of purified ornithine decarboxylase and amine compounds. 153 93


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