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)

The in vitro synthesis of elongation factor (EF)-Tu (tufB), the beta beta' subunits of RNA polymerase, ribosomal proteins L10 and L12 directed by DNA from the transducing phage lambda rifd 18, EF-Tu (tufA), EF-G, and the alpha subunit of RNA polymerase directed by DNA from the transducing phage lambda fus3 has been investigated in a crude and a partially defined protein-synthesizing system. Proteins L10 and L12 are synthesized in the partially defined system almost as well as in the crude system. However, the synthesis of EF-Tu, EF-G, and the alpha and beta beta' subunits of RNA polymerase is far less efficient in the partially defined system. An active fraction that stimulates the synthesis of these latter proteins has been obtained by fractionation of a high-speed supernatant on DEAE-cellulose. Because previous studies showed that this fraction (1 M DEAE salt eluate) contains a protein, called L factor, that stimulates beta-galactosidase synthesis in vitro, L factor was tested for activity. Although L factor stimulates the synthesis of the beta beta' subunits, it has little or no effect on the in vitro synthesis of the other products studied. In the present experiments, the ratio of L12/L10 and of EF-Tu (tufA)/EF-G formed is 4-6. These values are consistent with in vivo results.
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PMID:DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation. 16 May 61

When closed circular SV40 DNA containing 58 negative superhelical turns is used as a template for RNA synthesis with Escherichia coli RNA polymerase, a fraction of the RNA product remains complexed with the DNA. The RNA in the complex is resistant to ribonuclease in high salt, and the Tm indicates that it is hydrogen bonded to the DNA. The mole ratio of RNA to DNA nucleotides in the complex ranges from 0.01 to 0.08; the RNA ranges in length from 80 to 600 nucleotides. The formation of the complex is dependent on the circular DNA being topologically underwound since no complex is formed when closed circular DNA containing zero superhelical turns is used as the template. The DNA-RNA complex can serve as a primer-template combination for in vitro DNA synthesis by E. coli DNA polymerase I. After synthesis with (alpha-32P)-labeled deoxyribonucleoside triphosphates followed by alkaline hydrolysis, the isolation of 32P-labeled ribonucleotides is evidence for a covalent linkage between the RNA and the DNA synthesized. During the in vitro DNA synthesis, the template is nicked at a low rate, and the nicked molecules support extensive DNA synthesis. This observation indicates that only limited synthesis can occur on unnicked molecules possibly owing to the topological constraints against unwinding of the helix. Possible models for in vivo priming of double-stranded DNA by E. coli RNA polymerase are discussed.
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PMID:Priming of superhelical SV40 DNA by Escherichia coli RNA polymerase for in vitro DNA synthesis. 16 2

Cultures of the rat skeletal muscle myoblast cell line, L6, were treated with the mutagen ethylmethanesulfonate and grown in the presence of alpha-amanitin, an inhibitor of RNA polymerase II in vitro. One clonal cell line, Ama102, resistant tc the cytotoxic action of 2 mu-g/ml of alpha-amanitin was isolated and extensively characterized. Ama102 cells were about 30-fold more resistant to alpha-amanitin than their Ama+ parent cells based on a comparison of the concentration of alpha-amanitin required to reduce their plating efficiencies to similar extents. The RNA polymerase activities from Ama+ and Ama102 cells were solubilized and separated by DEAE-Sephadex chromatography. Whereas all of the Ama+ RNA polymerase II activity was inhibited by 0.1 mu-g/ml of alpha-amanitin, about 30% of the activity in the Ama102 RNA polymerase II peak was resistant to this concentration of alpha-amanitin and was inhibited only by much higher concentrations (25 mu-g/ml) of alpha-amanitin. This alpha-amanitin-resistant activity in Ama102 cells was identified as a bona fide RNA polymerase II by its chromatographic behavior on DEAE-Sephadex, salt optimum, preference for denatured DNA as template, insensitivity to inhibition by potassium phosphate, thermal inactivation kinetics, and inactivation by anti-RNA polymerase II antiserum. Both RNA polymerase IIa and IIb from Ama102 cells exhibited the partial alpha-amanitin resistance, as did this activity when purified further on phosphocellusose. Unlike the parental Ama+ cells, Ama102 cells neither fused at confluence nor showed an increase in the specific activity of creatine kinase. The altered sensitivity of the Ama102 RNA polymerase II to alpha-amanitin appears to account for the drug-resistant phenotype of these cells.
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PMID:Isolation and characterization of an alpha-amanitin-resistant rat myoblast mutant cell line possessing alpha-amanitin-resistant RNA polymerase II. 16 92

The [3H]estradiol exchange assay was used to characterize the nuclear estrogen receptor from the chick oviduct. After diethylstilibestrol (DES) treatment (14 days), the oviduct nuclei contained estrogen receptors that manifested high affinity (Kd = 2 X 10(-9)M) and low capacity binding (4000 to 5000 sites/cell) for estradiol. DES and estradiol competed significantly for [3H]estradiol binding, while testosterone and progesterone were ineffective. These binding sites were found in the oviduct and liver but not in the spleen, kidney, or muscle. Following salt extraction from nuclei, the receptor had a sedimentation coefficient of 4 S when analyzed by centrifugation in high and low salt sucrose density gradients. The [3H]estradiol exchange assay was used to examine the relationships between nuclear-bound receptor and RNA polymerase initiation sites on oviduct chromatin. Within 20 min after a single injection of 2.5 mg of DES to withdrawn chicks, a maximum number of estradiol receptor-binding sites was detected in oviduct nuclei. Within 30 min after DES injection, the total number of RNA initiation sites also increased, reaching 100% of control values. Daily injections of DES to unstimulated chicks resulted in a gradual increase in the nuclear content of estradiol receptor, which reached a maximum at 6 days and thereafter declined gradually up to 18 days of hormone treatment. A maximum number of initiation sites for RNA synthesis was also attained at 4 to 6 days of DES treatment and thereafter declined. When DES was withdrawn after 14 to 18 days of hormone stimulation, the numbers of nuclear estradiol receptor sites and initiation sites for RNA synthesis both declined gradually, reaching half-maximal values in 3 days and approached control values after 7 to 8 days of withdrawal. The increase in the concentration of nuclear estradiol receptor sites and the number of initiation sites for RNA synthesis also showed a close correlation with the dosage of DES administration. Both attained maximum levels at 1.25 mg of DES with a half-maximal value of 0.5 mg. The close correlation between the concentration of nuclear-bound estradiol receptors and the number of initiation sites for RNA synthesis in vivo is at present only a temporal correlation but does raise the possibility that gene transcription in chick oviduct may depend upon the amount of estradiol receptor bound to the target cell nuclei.
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PMID:Effect of estrogen on gene expression in the chick oviduct. Correlation between nuclear-bound estrogen receptor and chromatin initiation site for transcription. 17 20

DNA-dependent RNA polymerases (EC 2.7.7.6) were extracted and partially purified form the nuclei of rat ascites hepatoma cells (AH-130) induced by 4-dimethylaminoazobenzene. The patterns of RNA synthesis and the properties of these enzymes were compared with enzymes from the nuclei of rat liver. The specific activity of RNA polymerase in the homogenate from the nuclei of AH-130 cells was the same as normal rat liver nuclei. RNA polymerase was solubilized from the homogenate at high ionic strength and separated into two forms by DEAE-Sephadex column chromatography. Enzymatic characterization showed that these enzymes corresponded to RNA polymerase I and II. RNA polymerase I more effectively transcribed native DNA than denatured DNA at low salt concentration, but at high salt concentration RNA polymerase I effectively transcribed denatured DNA. RNA polymerase II more effectively transcribed denatured DNA. In AH-130 cells the activity of RNA polymerase I was 4 to 5 times higher than RNA polymerase II, and in rat liver the activity of RNA polymerase I was 1.5 to 2 times higher than RNA polymerase II. The activity of RNA polymerase I in AH-130 cells may have increased by induction.
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PMID:A comparative study of DNA-dependent RNA polymerases from rat ascites hepatoma cell nuclei and from rat liver nuclei. 18 Jul 54

Two forms of yeast RNA polymerase A are resolved by phosphocellulose chromatography. One of these, called RNA polymerase A, is lacking two polypeptide chains of 48,000 and 37,000 daltons. The properties of the two enzymes are compared in the present paper. RNA polymerase A transcribes d(A-T)n with a similar efficiency as the complete enzyme, but it is comparatively much less active with native DNA. The two enzymes can also be differentiated on the basis of their ionic strength and divalent cation requirements. RNA polymerase A has a particularly low activity at high salt and low Mg2+ concentrations. Thermal inactivation curves of the two enzymes are different when residual activity is assayed with native DNA. In contrast with d(A-T)n as template the apparent inactivation curves of the two enzymes are identical. The data suggest that the two dissociable polypeptide chains play an important role in transcription. The template specificity of yeast RNA polymerase B was further investigated using SV40 DNA-FI as template. RNA polymerase B is able to retain [3H]SV40 DNA-FI on nitrocellulose filters but the enzyme-DNA complex is very unstable. The observation that RNA polymerase B can transcribe to some extent a supercoiled DNA but not a linear double stranded template supports the hypothesis that the enzyme needs some unpaired DNA structure to initiate transcription.
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PMID:Further characterization of yeast RNA polymerases. Effect of subunits removal. 18 85

The strands of the polyoma genome coding for early and late viral RNA were separated by means of asymmetric cRNA synthesized under high salt conditions by Escherichia coli RNA polymerase. Each strand was then employed in RNA-DNA hybridization experiments to determine the degree to which it is transcribed in transformed cells under several conditions. Except for a concanavalin-A-selected revertant, approximately one-quarter of the early strand was expressed in all of the situations investigated. In contrast, while no significant late strand transcription was detected in transformed cells in culture, the tumors induced by these cells contained transcripts complementary to about 12% of this strand. The results are discussed in terms of current knowledge of the amount of the virus genome required to transform cells.
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PMID:Polyoma genome transcription in transformed mouse cells growing in culture and as tumors in syngeneic mice. 19 Jan 75

HSV-1 ANG DNA and a defective genome of the same virus were transcribed with E. coli RNA polymerase under various salt conditions. The extent of transcription was assayed by hybridizing the cRNA to the Hind III, Hpa I and Hind II restriction fragments of the DNA templates using the blot technique of E. Southern. The transcripts proved to contain sequences homologous to all DNA fragments. A similar ratio of hybridized cRNA and the amount of fragment DNA was observed in all cases. The results suggest that both, the wt and the defective HSV ANG genome were completely transcribed.
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PMID:In vitro transcription of herpes simplex virus ANG DNA by E-coli RNA polymerase. 19 92

The L and NS proteins of vesicular stomatitis virions (New Jersey serotype) were solubilized with Triton X-100 and high-salt buffer and recombined with purified nucleocapsids under conditions similar to those used to reconstitute transcriptase activity in vitro. The nucleocapsid-bound L and NS proteins were separated from unbound proteins on a glycerol gradient. The rebinding of L and NS proteins mimics the in vivo binding in that at saturation the ratio of L and NS molecules to N molecules is approximately the same as observed in the intact virion. L and NS proteins were separated and added back independently and in combination to the template. The purified NS protein bound to the template in the absence of L protein. However, the L protein binding appeared to depend on the presence of NS protein. The presence of Mg2+ and nucleotides, which is required for transcription, was not necessary for the rebinding of L and NS proteins.
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PMID:Rebinding of transcriptase components (L and NS proteins) to the nucleocapsid template of vesicular stomatitis virus. 21 81

We have found that nucleosomes reconstituted from histone octamers and SV40 DNA Form I by progressively decreasing the salt concentration from 2 M NaCl are formed preferentially around 0.27, 0.37, 0.50 and 0.85 on SV40 DNA (relative to the EcoRI site). When SV40 DNA Form III is used, the nucleosomes form mainly at 0.28, 0.38, 0.61 and 0.83. These sites are very close to both the sites of RNA chain initiation by calf thymus RNA polymerase B on SV40 DNA Form I (0.25, 0.35, 0.42 and 0.88) and the regions of the supercoiled DNA which are readily denaturable by T4 gene 32 protein (0.25, 0.47 and 0.88), and correspond to AT-rich regions as deduced from the nucleotide sequence of SV40 DNA. The physiologically important region around 0.67 is an unfavourable site for all three types of proteins, and corresponds to a GC-rich region surrounding a 17 base pair AT cluster.
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PMID:Preferential in vitro assembly of nucleosome cores on some AT-rich regions of SV40 DNA. 22 48


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