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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 kinetics of interaction of PPi and its diphosphonic analog, methylenediphosphonic acid (MDPA), with nucleoside triphosphates, DNA and Mg2+ binding sites of DNA-dependent RNA polymerase II from calf thymus was investigated. The values of apparent Km in the NTP polymerization reaction for ATP and CTP equal to 2.7 X 10(-4) and 1.8 X 10(-4) M, respectively, were determined. It was shown that MDPA and PPi competitively inhibited the RNA polymerase reaction with respect to nucleoside triphosphate. The inhibition constants (Ki) of ATP and CTP incorporation for MDPA were 2.2 X 10(-4) and 3.3 X 10(-4) M, respectively, while those of the nucleoside triphosphate incorporation for PPi were equal to 1.4 X 10(-4) and 2.0 X 10(-4) M, respectively. MDPA and PPi were incompetitive inhibitors of template (DNA) and Mn2+. A possible mechanism of inhibition of the RNA polymerase reaction by MDPA is proposed.
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PMID:[Kinetics of the interaction of methylene diphosphonic acid and inorganic pyrophosphate with DNA-dependent RNA-polymerase from calf thymus]. 298 49

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

The gene coding for CTP:CMP-3-deoxy-D-mannooctulosonate cytidylyltransferase (CMP-KDO synthetase), kds B, was previously cloned on a 9-kilobase Pst insert of Escherichia coli DNA into pBR 322 (Goldman, R. C., and Kohlbrenner, W. E. (1985) J. Bacteriol. 163, 256-261). Using a transposon mutagenesis approach we have now located kds B on this insert, which facilitated the isolation and sequencing of a 1.3-kilobase segment of DNA containing kds B and putative RNA polymerase and ribosome binding sites. The primary structure of CMP-KDO synthetase predicted by this nucleotide sequence was verified by amino acid composition and sequence analysis of purified CMP-KDO synthetase and cleavage fragments. Our results show that kds B consists of a 744-base open reading frame coding for a 248-amino acid peptide. The molecular weight of CMP-KDO synthetase calculated from the translated sequence is 27,486, taking into account the loss of the N-terminal methionine. These data define the transcriptional unit of kds B and its translation product in molecular terms, information prerequisite to our understanding of both the mechanism of CMP-KDO formation and the regulation of the KDO metabolic pathway in Gram-negative bacteria.
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PMID:Primary structure of CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) from Escherichia coli. 302 27

We have established conditions that stabilize the interaction between RNA polymerase and the rrnB P1 promoter in vitro. The requirements for quantitative complex formation are unusual for E. coli promoters: (1) The inclusion of a competitor is required to allow visualization of a specific footprint. (2) Low salt concentrations are necessary since complex formation is salt sensitive. (3) The addition of the initiating nucleotides ATP and CTP, resulting in a low rate of dinucleotide production, is required in order to prevent dissociation of the complexes. The complex has been examined using DNAase I footprinting and filter binding assays. It is characterized by a region protected from DNAase I cleavage that extends slightly upstream of the region protected by RNA polymerase in most E. coli promoters. We find that only one mole of active RNA polymerase is required per mole of promoter DNA in order to detect filter-bound complexes. Under the conditions measured, the rate of association of RNA polymerase with rrnB P1 is as rapid as, or more rapid than, that reported for any other E. coli or bacteriophage promoter.
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PMID:Visualization and quantitative analysis of complex formation between E. coli RNA polymerase and an rRNA promoter in vitro. 305 11

We investigated the transcription kinetics of RNA polymerase from an rpoBC mutant of Salmonella typhimurium which showed highly elevated, constitutive expression of the pyrB and pyrE genes as well as an increased cellular pool of UTP. When bacterial cultures containing an F' lac+ episome were induced for lac operon expression, the first active molecules of beta-galactosidase were formed with a delay of 73 +/- 3 s in rpo+ cells. The corresponding time was 104 to 125 s for cells carrying the rpoBC allele, indicating that this mutation causes a reduced RNA chain growth rate. In vitro the purified mutant RNA polymerase elongated transcripts of both T7 DNA and synthetic templates more slowly than the parental enzyme at a given concentration of nucleoside triphosphates. This defect was found to result from four- to sixfold-higher Km values for the saturation of the elongation site by ATP and UTP. The saturation kinetics of the RNA chain initiation step also seemed to be affected. The maximal elongation rate and Km for GTP and CTP were less influenced by the rpoBC mutation. Open complex formation at the promoters of T7 DNA and termination of the 7,100-nucleotide transcript showed no significant difference between the parental and mutant enzymes. Together with the phenotype of the rpoBC mutant, these results indicate that expression of pyrB and pyrE is regulated by the mRNA chain growth rate, which is controlled by the cellular UTP pool. The rate of gene expression is high when the saturation of RNA polymerase with UTP is low and vice versa.
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PMID:Association of RNA polymerase having increased Km for ATP and UTP with hyperexpression of the pyrB and pyrE genes of Salmonella typhimurium. 308 91

Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.
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PMID:Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates. 313 51

Wheat germ RNA polymerase II is able to transcribe polynucleotide templates in the poly-[d(G-C)] family, adopting either the right-handed B or left-handed Z conformations depending on the ionic environment and temperature. Thus, with poly[d(G-C)] either the B state (in MgCl2) or the associated Z* state (in MnCl2) can be established. Poly[d(G-m5C)] adopts the Z form readily in MgCl2, and poly-[d(G-br5C)] can be regarded as being "constitutively" in the Z state. In transcription studies with CpG as a primer and templates in the left-handed conformation, it is found that the rate of productive elongation, i.e., the synthesis of poly[r(G-C)], is depressed, in accordance with the results of previous studies. However, with a single triphosphate substrate, CTP, the rate of formation of the first phosphodiester bond, i.e., the synthesis of CpGpC, is about 4-fold greater with both the Z and Z* templates than with B-DNA. This transcriptional activity is also catalytic in the sense that product concentrations exceed that of the enzyme. The synthesis of CpGpC is reduced in the presence of GTP. However, the apparent Km value for GTP utilization is lower for the trinucleotide synthesis (0.1 microM) than that obtained for productive elongation (0.8 microM), a result that also holds for B-DNA templates. All transcription reactions are specifically inhibited by the fungal toxin alpha-amanitin, and, in the case of the left-handed templates, by monoclonal anti-Z-DNA antibodies. The relative probabilities of single-step addition and productive elongation imply that the major distinction between transcription of templates in the B and Z conformations involves a step following the synthesis of the first phosphodiester bond. As a result, fully competent elongation complexes do not form on the left-handed DNA.
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PMID:Transcription of left-handed Z-DNA templates: increased rate of single-step addition reactions catalyzed by wheat germ RNA polymerase II. 321 41

The complex [promoter A2 X E. coli RNA polymerase] was treated with phosphoamides, derivatives of 4-[N-methyl, N-(2-chloroethyl)]-aminobenzylamine and guanosine-5'-mono-, di-, and triphosphates with the alkylating group attached to the terminal phosphates. After this, [alpha-32P]CTP was added. Residues of the affinity reagents bound covalently at the first stage were elongated by radioactive -pC residues due to the catalytic action of the active centre of RNA polymerase. Affinity labelled were beta-and sigma-subunits of the enzyme, and the promoter. The affinity label was localized on -pGpC residues. A guanine residue was alkylated in the promoter as suggested by radioactivity elimination kinetics. As the data obtained and the previously known length of the reagent (maximum distance between the alpha-phosphorus atom of the reagent and the point of alkylation is less than 0.6 nm) indicate, there is a direct rather than protein-mediated contact between the template and the substrate within the complex [promoter X RNA polymerase].
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PMID:[Highly selective affinity labeling of a promoter in a complex with E. coli RNA-polymerase by alkylating derivatives of initiating substrates]. 330 Jun 59

An in vitro system for the synthesis of influenza viral RNA was developed using isolated nuclei prepared from influenza virus-infected HeLa cells. In this system, two species of positive-sense RNA, i.e., mRNA and cRNA (complementary RNA to vRNA), were found to be synthesized when analyzed by RNA-RNA hybridization using a minus-strand RNA probe and high resolution gel electrophoresis. The in vitro RNA synthesis required Mg2+, GTP, CTP, UTP, a high concentration of ATP, and an ATP regenerating system. Neither actinomycin D nor alpha-amanitin, potent inhibitors for cellular DNA-dependent RNA polymerases, inhibited the RNA synthesis. Addition of ApG or capped RNA, well-known primers for virion-associated RNA polymerase, markedly enhanced the extent of RNA synthesis. The maximum activity was observed with nuclei isolated from cells at 5 h after infection. This system is useful for the purification and characterization of factors involved in the transcription of these two species positive-sense RNA.
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PMID:In vitro synthesis of influenza viral RNA: characterization of an isolated nuclear system that supports transcription of influenza viral RNA. 361 Oct 43

DNA primase (EC 2.7.7.6) produces an RNA oligomer of approximately 10 bases, which is required by DNA polymerase alpha (EC 2.7.7.7) for the initiation of DNA synthesis. We partially purified DNA primase from acute lymphocytic leukemia cells from patients using several chromatography columns. Poly(dT) and poly(dC), but not poly(dA) or poly(dG), were good templates for ribonucleoside triphosphate (rNTP)-dependent DNA synthesis (i.e., DNA primase activity), and they were used in the study of the effect of natural and arabinofuranosyl nucleoside triphosphates on DNA primase activity. The Km for GTP in the poly(dC) primase assay was approximately 175 microM. All noncomplementary natural rNTPs and deoxyribonucleoside triphosphates (dNTPs) inhibited poly(dC) primase activity to a similar extent (Ki values of ATP and CTP were 610 and 517 microM, respectively). 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) and 9-beta-D-arabinofuranosyladenine 5'-triphosphate (araATP) were more potent inhibitors of poly(dC) primase activity than were CTP and ATP (Ki values were approximately 125 microM). araCTP, araATP, CTP, and ATP inhibited DNA primase activity in a manner competitive with GTP. The concentration required to inhibit poly(dC) DNA primase activity by 50% was determined for a number of arabinofuranosyl nucleoside triphosphate analogs, and the relative potency of inhibition of DNA primase activity was as follows: rNTP = dNTP = 5-aza-dCTP less than ara-5-azaCTP = araTTP = araATP = araCTP less than 2-fluoro-araATP = 2'-azido-2'-deoxy araCTP less than 2'-fluoro-araTTP = 2'-fluoro-5-iodo-araCTP = 2'-fluoro-5-methyl-araCTP. In the poly(dT) primase assay ATP did not follow classic Michaelis-Menten kinetics (ATP exhibited positive cooperativity with a Hill coefficient of 2.0). However, this assay was very sensitive to araCTP (apparent Ki of 25 microM). In summary, these experiments suggested that DNA primase is controlled by the levels of ribonucleoside triphosphates, and that the perturbation of these pools by any agent could lead to the inhibition of DNA primase and thereby inhibit DNA synthesis. Furthermore, aranucleoside triphosphate analogs directly inhibited DNA primase, and it is possible that this effect may contribute to the cytotoxicity of these compounds.
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PMID:Inhibition of DNA primase by nucleoside triphosphates and their arabinofuranosyl analogs. 380 92


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