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)

A factor stimulating RNA polymerase II from Ehrlich ascites tumor cells was purified. The final preparation appeared almost homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 38 000. The endonuclease activity of about 10 mug of purified factor, if any was well below the 10(-5) mug equivalent of pancreatic deoxyribonuclease, indicating that the stimulation of RNA synthesis by this factor was not due to contaminating endonuclease. This factor specifically stimulated RNA polymerase II on native DNA as template and did not affect RNA polymerase I at all. The molecular size of RNA synthesized in the presence of this factor increased markedly compared with that synthetized by RNA polymerase II alone.
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PMID:Purification of a factor from Ehrlich ascites tumor cells specifically stimulating RNA polymerase II. 99 Feb 65

DNA-dependent RNA polymerase I (or A) was purified from rat liver nucleoli. DNA was effectively removed from the solubilized enzyme with a defined concentration of polyethyleneglycol. The enzyme was purified further with successive DEAE-Sephadex and phosphocellulose column chromatography followed by glycerol gradient centrifugation. The procedure yielded an electrophoretically homogeneous enzyme with a specific activity 400 times that of the nucleolar extracts. The recovery of the activity was approximately 20%. The RNA polymerase I eluted as a single peak from DEAE-Sephadex was separated into two distinct peaks by a phosphocellulose column. The first peak eluting at about 0.12 M ammonium sulfate was designated as RNA polymerase IA and the second peak eluting at about 0.18 M as RNA polymerase IB. In normal rat liver nucleoli IA enzyme comprised approximately 20% of the total RNA polymerase I activity and the IB enzyme comprised approximately 80%. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, enzyme IB contained five subunits with molecular weights of 195000 (a), 130000 (b), 65000 (c), 40000 (d), and 19000 (e) at nearly equimolar amounts. The calculated molecular weight of the enzyme (449000) agreed well with that predicted from the sedimentation coefficient of the enzyme. Enzyme IA contained identical subunits except that subunit c was absent. Preliminary studies could not demonstrate any significant differences in template specificity between IA and IB enzyme.
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PMID:Nucleolar DNA-dependent RNA polymerase from rat liver. 1. Purification and subunit structure. 100 57

Bacteriphage T7 RNAs have been fractionated on preparative polyacrylamide gels. The in vitro coding capacities of the RNAs have been determined by translation of the RNAs in a cell-free system and analysis of the polypeptide products on sodium dodecyl sulfate polyacrylamide slab gels. The T7 early RNAs are fractionated according to their molecular weight and without intermolecular aggregation. Fractionation of the late T7 RNAs gives rise to 10 major RNAs, ranging in size from 0.29 X 10(6) daltons to 2.05 X 10(6) daltons. Five of these RNAs are polycistronic and overlapping species are present for some T7 proteins. In particular, the gene 10 protein, the major capsid protein, is translated from at least three mRNAs. The smallest of these gene 10 mRNAs is monocistronic. A second gene 10 mRNA also codes for the gene 9 protein, and a third gene 10 mRNA codes for both gene 8 and gene 9 proteins. The T7 gene 3.5 protein, a T7 lytic enzyme, is also translated from several differently sized mRNAs. Comparison with published data on in vitro transcription by T7 RNA polymerase suggestes that transcription from multiple initiation sites and cleavage of larger precursors are both involved in generating the late T7 transcripts we observe. The overlapping mode of transcription could serve to amplify certain gene products.
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PMID:Detection of polycistronic and overlapping bacteriophage T7 late transcripts by in vitro translation. 106 Nov 35

3-Ethoxy-8-methyl-5,6-dihydro-7H-cyclopenta[f]isoquinolin-5-one (2) was converted to 6-carbethoxymethyl-3-ethoxy-8-methyl-5,6-dihydro-7H-cyclopenta[f]isoquinolin-5-one (6) through an oxalyl derivative. Treatment of 6 with ammonia gave the corresponding amide 7 which on sodium borohydride reduction and subsequent dehydration yielded 6-carbamylmethyl-3-ethoxy-8-methyl-7(5)H-cyclopenta[f]isoquinoline (9). The analogous ester 10 was similarly obtained from 6. Numerous attempts to dealkylate the 3-ethoxy group of 9 or 10 failed. However, 6 coould easily be dealkylated on heating with 25% hydrochloric acid in a sealed tube.The ester, 6-carbethoxymethyl-8-methyl-5,6-dihydro-7H-cyclopenta[f]isoquinoline-3(2H),5-dione (11), so obtained was converted to the corresponding amide 12 which on reduction with sodium borohydride and subsequent dehydration afforded the desired compound, 6-car-bamylmethyl-8-methyl-7(5)H-cyclopental[f]isoquinolin-3-(2H)-one (1). 1 was found to be mildly cytotoxic againstL5178Y mouse leukemia cells in culture.1 was also found to bind to native calf thymus DNA. 1 inhibited RNA synthesis by a DNA-dependent RNA polymerase and a higher inhibition of RNA synthesis was observed when poly(dG-dC) was used as a template than when poly(dA-dT) was used. A significant increase of thermal transition temperature of calf thymus DNA and poly(dG)-poly(dC) was observed in the presence of 1. The accumulated evidence demonstrates that 1 interacts weakly with calf thymus DNA and interacts preferentially with poly(deoxyribonucleotides)-containing GC pairs.
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PMID:Cyclopenta[f]isoquinoline derivatives designed to bind specifically to native deoxyribonucleic acid. 2. Synthesis of 6-carbamylmethyl-8-methyl-7(5)H-cyclopenta[f]isoquinolin-3(2H)-one and its interaction with deoxyribonucleic acids and poly(deoxyribonucleotides). 109 35

An improved method is described for the purification of the DNA-dependent RNA polymerase [ribonucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6] from Escherichia coli. The method involves lysozyme-sodium deoxycholate lysis, low-speed centrifugation, precipitation with Polymin P, elution from the Polymin P precipitate, ammonium sulfate precipitation, and chromatography on DNA-cellulose and Bio-Gel A 5m. RNA polymerase is purified to electrophoretic homogeneity in 2 days with a recovery of 45%, resulting in a yield of 250 mg of holoenzyme from 500 g of cells.
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PMID:A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. 110 52

A soluble extract prepared from T7-infected E. coli is able to initiate DNA synthesis on an exogenous T7 DNA template. We have developed a fractionation procedure to resolve and identify the proteins required for T7 DNA synthesis. By this method we have purified the following T7 replication-related proteins (each greater than 50% pure as judged by sodium dodecyl sulfate gel electrophoresis): T7 DNA-binding protein (27,000 daltons), T7 RNA polymerase (105,000 daltons), T7 DNA polymerase (gene 5-protein, 85,000 daltons, plus host-factor), T7 DNA ligase (40,000 daltons), and T7 DNA-priming protein (65,000 daltons). The T7 DNA-priming protein, synthesized between 7.5 and 15 min following infection, was not detectable if the infecting phage carried an amber mutation in gene 4. Using an in vitro complementation assay which specifically measures the stimulation of DNA synthesis in an extract prepared from T7 gene 4-mutant infected cells, we have purified the DNA-priming protein about 2,000-fold. The purified priming protein preparations are essentially free of endonuclease, exonuclease, DNA ligase and DNA polymerase activity, but they do contain measurable DNA-dependent RNA synthetic acitvity. The enzyme is rapidly inactivated by heating to 46 degrees C and by treatment with N-ethylmalemide. In the presence of T7 DNA-binding protein and all four ribonucleoside triphosphates, the DNA-priming protein enables T7 DNA polymerase to initiate DNA synthesis on intact duplex T7 DNA. Closer studies of its enzymatic function as well as of the possible roles of the other proteins in the T7 replication system will be presented in the accompanying paper.
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PMID:Studies on bacteriophage T7 DNA synthesis in vitro. I. Resolution of the T7 replication system into its components. 110 17

Bovine lymphosarcoma tissue has been extracted with low- and high-salt buffers [0.05 M Tris-C1 plus or minus 0.3 M (NH4) 2S04]. Diethylaminoethyl-Sephadex chromatography of both the high-salt and low-salt extracts yields RNA polymerases I and II, although low-salt extraction releases only one-third as much activity. Extraction by high salt of the residue from the low-salt extract, followed by diethylaminoethyl-Sephadex chromatography, yields additional enzyme activity with properties of Form II. Purification of the low-salt extract by protamine precipitation, elution with sodium succinate, and phosphocellulose chromatography yields a preparation of RNA polymerase (RNAP) with hybrid properties, combining the salt optimum of Form I, diethylaminoethyl-Sephadex elution pattern of form II, and alpha-amanitin sensitivity of Form III. RNAP. transcribes native D,A and chromatin efficiently. More RNAPL is recovered from lymphosarcoma tissue than from calf thymus.
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PMID:RNA polymerase isolated from bovine lymphosarcoma by sequential low- and high-salt extraction. 117 19

A non-histone protein has been isolated from Ehrlich ascites tumor chromatin. The minimum molecular weight of this non-histone protein, estimated by sodium dodecyl sulfate gel electrophoresis and amino acid analysis, is approximately 10 to 11,000. This non-histone protein is acidic, contains 2.7% alkalilabile phosphorus, binds to DNA, and inhibits transcription of DNA in vitro by the homologous RNA polymerase. The per cent inhibition of RNA synthesis is not affected by increasing amounts of RNA polymerase, but is reduced by addition of excess DNA. In the presence of the non-histone protein, incorporation of [gamma-32P]ATP into RNA in the in vitro RNA synthesizing system is inhibited, with no apparent change in the average chain length of the RNA product. Inhibition of RNA synthesis is completely eliminated if the DNA template is allowed to interact with ATP prior to the addition of the non-histone protein. These results indicate that the observed repression of in vitro RNA synthesis is due to the effect of the non-histone protein on the DNA, inhibiting the initiation of RNA chain formation.
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PMID:Inhibition of transcription in vitro by a non-histone protein isolated from Ehrlich ascites tumor chromatin. 119 69

The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.
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PMID:Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells. 130 73

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.
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PMID:Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli. 130 92


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