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 influenza virus-associated RNA polymerase cleaves capped RNA in an endonucleolytic manner and the transcription is initiated by the addition of GMP, the first substrate to be polymerized under the direction of viral RNA template, onto 3'-termini of resulting capped RNA fragments. In the presence of high concentrations of GTP as a sole substrate, multiple GMP residues were polymerized onto the primers. By the addition of the second substrate CTP, excess GMP residues, other than the 1st residue, were removed prior to elongation. The result may suggest that the RNA-dependent RNA polymerase carries a proofreading function.
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PMID:Proofreading function associated with the RNA-dependent RNA polymerase from influenza virus. 301 29

A new Escherichia coli RNA polymerase mutant was isolated which exhibited reduced accuracy of chain elongation in vivo and in vitro. The novel isolation procedure consisted of simultaneous selection for rifampicin resistance and screening for increased leakiness of an early, strongly polar nonsense mutation of lacZ, one of a special class of mutations whose leakiness reflects mainly transcriptional rather than translational errors. The spontaneous mutant thus isolated displayed a 3-4-fold increase in the leakiness of two different lacZ mutations of this class. Transduction analysis indicated that a single mutation, mapping in or very near the rpoB gene for the beta subunit of RNA polymerase, conferred both rifampicin resistance and increased nonsense leakiness. In an in vitro fidelity assay, homogeneous RNA polymerases from the mutant and parent strains exhibited error rates of 1/0.90 X 10(5) and 1/2.0 X 10(5), respectively, for the poly[d(A-T)] X poly[d(A-T)]-directed misincorporation of noncomplementary GMP. These error rates were verified by product analyses which further revealed that GMP was misincorporated in place of AMP in the synthesis of poly[r(A-U)]. The error rate of wild-type K12 RNA polymerase from a different source was 1/2.0 X 10(5), while that of a hybrid RNA polymerase, containing mutant core enzyme and wild-type sigma subunit, was 1/0.64 X 10(5). These error rates confirmed the selection of a transcriptional accuracy mutant. The error frequencies observed are much lower than those reported in other in vitro assays. The safeguards used to avoid artifactually enhanced misincorporation, and to thereby quantitate lower error rates, are discussed.
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PMID:An RNA polymerase mutant with reduced accuracy of chain elongation. 309 80

Superselective affinity labelling of E. coli RNA polymerase in a complex with the promoter-containing fragment of T7 DNA by treatment with orto-formylphenyl ester of GMP followed by addition of [alpha-33P]UTP resulted in covalent binding of the residue--pGpU (p-radioactive phosphate) with one of lysine residues of the beta-subunit, Lys1048, Lys1051, Lys1057, Lys1065. The amino acid sequence of this region of the beta-subunit of E. coli RNA polymerase has a high extent of homology with that deduced for a region of tobacco chloroplast RNA polymerase on the basis of the nucleotide sequence of the chloroplast rpoB-like gene.
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PMID:[Localization of lysine residues in the site of initiating substrate binding of E. coli RNA-polymerase]. 311 88

Immediately following initiation of transcription, T7 RNA polymerase enters a phase in which dissociation of the enzyme-DNA-RNA ternary complex significantly competes with elongation, a process referred to in the Escherichia coli enzyme as abortive cycling [Carpousis, A.J., & Gralla, J.D. (1980) Biochemistry 19, 3245-3253]. Characterization of this process in the T7 RNA polymerase system under various reaction conditions and on templates with differing message sequences reveals that conversion to a highly processive ternary complex occurs after incorporation of eight bases and that the relative competition between dissociation and elongation up to this point is influenced by several different forces. In particular, the sequence dependence of abortive falloff suggests that dissociation is favored immediately following incorporation of UMP and is less likely following incorporation of GMP into the RNA message. Abortive cycling is unchanged in transcription from a synthetic oligonucleotide template which is double-stranded in the promoter region but single-stranded throughout the entire message region. This result proves that melting and reannealing of the DNA duplex in the coding region do not contribute to abortive cycling. Furthermore, weakening of promoter binding by an order of magnitude affects abortive cycling only slightly, suggesting that strong interactions with the promoter are not the major cause of abortive cycling. Kinetic analyses show that conversion to a highly processive ternary complex after the incorporation of eight bases may reflect a large decrease in the unimolecular rate of dissociation of the complex due to increased contacts between the nascent RNA and the DNA template and between RNA and enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Processivity in early stages of transcription by T7 RNA polymerase. 341 67

Nuclear protein factor type 1 (NPF-1) that simulates IMR-32 primase-associated DNA polymerase alpha 1 and alpha 2 activities has been purified from a high-salt extract of liver chromatin from 6-month-old rats. The final purified factor lacks DNA polymerase alpha, RNA polymerase, and DNA-unwinding or topoisomerase type I activities. The stimulatory activity is destroyed by trypsin (60 min at 37 degrees C), DNase II (60 min at 37 degrees C), and heat treatment (2 min at 68 degrees C). The 125I-labeled NPF-1 does not bind to activated calf thymus DNA or poly(dC). However, it forms a ternary complex with DNA in the presence of DNA polymerase alpha-primase complex (alpha 1 and alpha 2). The ternary complex sediments on sucrose density gradient as a heavier band (11S). The NPF-1 also stimulates (2.5-fold) primase-catalyzed incorporation of GMP and dGMP from the corresponding triphosphates on poly(dC) template even in the presence of a high concentration of alpha-amanitin (400 micrograms/ml). The labeled duplex containing the poly(dC) template, [32P]-GTP, and [3H]dGTP loses 80% of the 32P label and 70% of the 3H label after treatment with 0.3 M KOH and DNase I, respectively. The products were isolated from reaction mixtures incubated with and without NPF-1 and subjected to alkaline sucrose-density-gradient sedimentation analysis. The results suggest that the rate of synthesis of DNA short chains is increased in the presence of NPF-1 without a concomitant increase in the chain length of the newly synthesized products.
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PMID:Stimulation of human neuroblastoma DNA polymerase alpha and primase activities by a protein factor isolated from rat liver chromatin. 354 Sep 37

An RNA polymerase-viral RNA complex was purified from influenza A/PR/8 virions by combination of cesium trifluoroacetate centrifugation and phosphocellulose column chromatography. Surface proteins were removed from the detergent-treated virions by the centrifugation. Starting from the M protein-free ribonucleoprotein (RNP) fraction, an RNA polymerase-RNA complex lacking NP protein was isolated by repeated chromatography on phosphocellulose columns. The isolated RNA polymerase-RNA complex, which is composed of PB1, PB2, PA and vRNA, cleaved capped poly(A) endonucleolytically at 10-12 nucleotides from the 5' end and incorporated GMP into the 3' end of the resulting capped fragments. In the presence of all four ribonucleotide triphosphate substrates, the cleaved fragments were elongated to polynucleotides in the absence of exogenous vRNA. The RNA synthesis was primed not only by capped polynucleotides but also dinucleotide ApG. These results indicate that the purified RNA polymerase-RNA complex is as active in viral mRNAs synthesis as native RNP and that NP protein is not required for the catalytic function.
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PMID:Purification and enzymatic properties of an RNA polymerase-RNA complex from influenza virus. 384 Jun 35

Exogenous polyribonucleotides stimulated the ribonucleic acid (RNA) transcriptase in Sendai virions. Added yeast RNA, polyadenylic acid, or polycytidylic acid increased incorporation of (3)H-guanosine monophosphate as much as fivefold. The products of stimulated reactions were virus-specific as determined by hybridization with Sendai virion RNA, but they sedimented more slowly (13s) than the product of an unstimulated reaction (16s). The stimulating activity was nondialyzable and heat stable, but was abolished by alkaline hydrolysis. Nucleoside monophosphates, individually or in combination, were ineffective, confirming the requirement for a polymer. Among other substances tested for effects on Sendai virion transcriptase, polyaspartic acid and polyglutamic acid stimulated the enzyme; polyinosinic acid, polyuridylic acid, and polyamines had no effect; and dextran sulfate and polyvinyl sulfate were inhibitory.
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PMID:Stimulation of Sendai virion transcriptase by polyanions. 434 27

1. The interaction of aflatoxin B(1) with different polynucleotides was studied spectrophotometrically. Equations were derived that enable the degree of binding to be determined without first determining the extinction coefficient of the bound form. 2. The interaction with calf thymus DNA obeys first-order relationships with an association constant of 0.40mm(-1), but there is some evidence for a secondary binding process from results obtained at 390nm. 3. The spectral shifts decreased in the order polyadenylic acid+polyuridylic acid>DNA>polyadenylic acid>polyadenylic acid+polyinosinic acid. Polycytidylic acid, polyuridylic acid, polyinosinic acid (both single- and triple-stranded), AMP, CMP, GMP and UMP did not interact with aflatoxin. It was concluded that there is a requirement for the amino group of adenine (or possibly guanine) for binding of aflatoxin to polynucleotides to occur. 4. Binding is reversed by increasing ionic strength, and by Mn(2+) and Mg(2+) in the concentration range studied (0-5mm). The effect of the Mn(2+) or Mg(2+) was far greater than would be expected on the basis of their ionic strength. With both the bivalent cations and sodium chloride the reversal is greatest with double-stranded polynucleotides. 5. Inhibition in vitro of the DNA-dependent RNA polymerase of Escherichia coli by aflatoxin B(1) was detected only in the absence of Mg(2+) and at concentrations of Mn(2+) below the optimum for RNA synthesis in vitro. 6. The degree of inhibition (maximally 30%) was dependent on the concentration of Mn(2+) and decreased during incubation.
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PMID:The interaction of aflatoxin B1 with polynucleotides and its effect on ribonucleic acid polymerase. 489 17

1. The Widnell & Tata (1966) assay method for Mg(2+)-activated DNA-dependent RNA polymerase was used for initial-velocity determinations of rat liver nuclear RNA polymerase. One unit (U) of RNA polymerase was defined as that amount of enzyme required for 1 mmol of [(3)H]GMP incorporation/min at 37 degrees C. 2. Colony fed rats were found to have a mean RNA polymerase activity of 65.9muU/mg of DNA and 18h-starved rats had a mean activity of 53.2muU/mg of DNA. Longer periods of starvation did not significantly decrease RNA polymerase activity further. 3. Rats that had been starved for 18h were used for all feeding experiments. Complete and tryptophan-deficient amino acid mixtures were given by stomach tube and the animals were killed 15-120min later. The response of RNA polymerase to the feeding with the complete amino acid mixture was rapid and almost linear over the first hour of feeding, resulting in a doubling of activity. The activity was still elevated above the starvation value at 120min after feeding. The tryptophan-deficient amino acid mixture produced a much less vigorous response about 45min after the feeding, and the activity had returned to the starvation value by 120min after the feeding. 4. The response of RNA polymerase to the feeding with the complete amino acid mixture was shown to occur within a period of less than 5min to about 10min after the feeding. 5. Pretreatment of the animals with puromycin or cycloheximide was found to abolish the 15min RNA polymerase response to the feeding with the complete amino acid mixture, but the activity of the controls was unaffected. 6. The characteristics of the RNA polymerase from 18h-starved animals and animals fed with the complete or incomplete amino acid mixtures for 1h were examined. The effects of Mg(2+) ions, pH, actinomycin D and nucleoside triphosphate omissions were determined. The [Mg(2+)]- and pH-activity profiles of the RNA polymerase from the animal fed with the complete mixture appeared to differ from those of the enzyme from the other groups, but this difference is probably not significant. 7. [5-(3)H]Orotic acid incorporation by rat liver nuclei in vivo was shown to be affected by the amino acid mixtures in a similar manner to the RNA polymerase. 8. The tryptophan concentrations of plasma and liver were determined up to 120 min after feeding with the amino acid mixtures. Feeding with the complete mixture produced a rapid increase in free tryptophan concentrations in both plasma and liver, but feeding with the incomplete mixture did not alter the plasma concentration. The liver tryptophan concentration increased at about 45min after feeding with the tryptophan-deficient diet. 9. There was a good correlation between the liver tryptophan concentration and RNA polymerase activity in all groups of animals. 10. It was concluded that the rat liver nucleus responded to an increase in amino acid supply by increased synthesis of RNA as a result of synthesis of RNA polymerase de novo. The correlation of tryptophan concentration and RNA polymerase activity appears to reflect the general amino acid concentration required to support hepatic protein synthesis and to produce new RNA polymerase. This new polymerase appears to differ from the basal RNA polymerase by its rapid synthesis and destruction, which may be a means of regulating RNA synthesis by the amino acid concentration in the liver.
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PMID:The effect of feeding with a tryptophan-free amino acid mixture on rat liver magnesium ion-activated deoxyribonucleic acid-dependent ribonucleic acid polymerase. 549 25

On the basis of our observation of the increased specific activities of glutamine-utilizing enzymes in purine and pyrimidine metabolism in hepatoma 3924A, and because the concentration of glutamine is ten times lower in the hepatomas than in the liver, the biochemical pharmacology of the anti-glutamine agent, acivicin, was examined. (1) Acivicin competitively inhibited the activities of amidophosphoribosyl-transferase, CTP synthetase and carbamoyl-phosphate synthetase II from extracts of liver and hepatoma 3924A. (2) In addition to the competitive inhibition exerted by acivicin, evidence was obtained that this drug also irreversibly inactivated in vitro the glutamine-utilizing enzymes. It is particularly relevant for the selectivity of acivicin that the activity of aspartate carbamoyltransferase, an enzyme present in the same complex as carbamoyl-phosphate synthetase II, was not affected by the anti-glutamine agent. (3) Acivicin in vivo brought down the activities of glutamine-utilizing enzymes in a period of 10 min to 1 hr after injection. CTP synthetase activity declined to less than 10% of that observed in the uninjected rats. The decreases were not reversible by various in vitro methods, but in vivo the activities returned to normal range in 72 hr. (4) The activity of aspartate carbamoyltransferase, which exists as a multi-enzyme complex with synthetase II, was not altered by acivicin injection. Similar results were observed in transplantable sarcoma in the rat. (5) The acivicin-induced decrease in enzymic activities could not be restored by purification of the enzymes. (6) In vitro studies indicated that addition of acivicin to liver or hepatoma extracts or purified enzymes rapidly decreased enzymic activities; the activities could not be restored. These results are consistent with an interpretation that acivicin acts either as a tight-binding inhibitor or as an inactivator through alkylation of the enzymes of glutamine utilization. (7) Acivicin in combination with actinomycin provided a synergistic kill of hepatoma cells in tissue culture and also inhibited the growth of transplantable solid hepatoma 3924A in the rat. (8) The synergistic biological results of combination chemotherapy with acivicin and actinomycin can be accounted for by the action of acivicin in inhibiting GMP and CTP synthetases, resulting in a decrease in GTP and CTP content, and by the actinomycin-caused inhibition of RNA polymerase in selectively blocking the utilization of GTP and CTP.
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PMID:Multi-enzyme-targeted chemotherapy by acivicin and actinomycin. 618 Jun 9


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