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)

Mammalian cells are known to synthesize DNA in discrete stages, the first of which seems to be the formation of DNA pieces 150--200 nucleotides in length that have a s20 value of about 4 S. We have reconstructed a system derived from HeLa cell nuclei that carries out RNA-primed initiation of the synthesis of small (4S) DNA fragments. This synthesis is resistant to high concentrations of alpha-amanitin and sensitive to antibody directed against RNA polymerase I, suggesting that this enzyme may be involved in the initiation step. The formation of small DNA fragments in this system also requires DNA polymerase alpha, heat-labile nuclear factor(s), and at least one other nuclear protein.
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PMID:Initiation of HeLa cell DNA synthesis in a subnuclear system. 28 14

A single peak of DNA polymerase activity was detectable by phosphocellulose chromatography of leukemic guinea pig lymphoblast whole cell extracts. The inability to detect multiple peaks of activity as described with other cell types is shown to be due to the insolubility of a large proportion of the DNA polymerase activity under the extraction condition used. Multiple forms of DNA polymerase with different template specificities were recognized in extracts of the subcellular fractions of these cells after chromatography on phosphocellulose and DEAE cellulose. On Sephadex G-200 gel filtration these enzymes had apparent molecular weights in excess of 140,000 daltons. No RNA polymerase (reverse transcriptase) was detected in any subcellular fraction despite the presence of oncornavirus like particles in these cells.
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PMID:Studies of the template preference and other characteristics of the DNA polymerases of leukemic guinea pig lymphoblasts. 29

Purified yeast DNA was transcribed by homologous RNA polymerases I and II and Escherichia coli RNA polymerase. Transcripts synthesized in vitro were analyzed by molecular hybridization with complementary DNA (cDNA) synthesized from yeast poly(A)-containing mRNA with viral reverse transcriptase and ribosomal DNA labeled in vitro by nick translation with E. coli DNA polymerase I. RNA synthesized by polymerase I and II in the presence of Mn2+ contained sequences complementary to cDNA and rDNA at a frequency consistent with random transcription of the template. Similarly, E. coli RNA polymerase synthesized an apparently random transcript in the presence of either Mn2+ or Mg2+. In contrast to these results, RNA polymerase I but not polymerase II transcripts were markedly enriched in sequences complementary to rDNA when transcription was carried out in the presence of Mg2+. The observed enrichment was 15-30-fold higher than observed for polymerase II or E. coli polymerase transcripts and is consistent with the transcript being comprised of 6-10% ribosomal sequences. These data strongly suggest that RNA polymerase I plays a critical role in selective transcription of ribosomal cistrons.
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PMID:Transcription of yeast DNA by homologous RNA polymerases I and II: selective transcription of ribosomal genes by RNA polymerase I. 31 52

In the presence of RNA polymerase, RNase H, discriminatory factors alpha and beta, Escherichia coli binding protein, DNA elongation factor I, DNA elongation factor II preparation, DNA polymerase III, and ATP, UTP, GTP, CTP, dATP, dTTP, dGTP, and dCTP, fd viral DNA can be quantitatively converted to RFII containing a unique gap in the linear minus strand. This gap, mapped with the aid of restriction endonucleases HinII and HpaII, is located within Fragment Hpa-H of the fd genome. The discrimination reaction has been resolved into two steps: Step A, fd viral DNA, E. coli binding protein, and discriminatory factors alpha and beta form a protein DNA complex; Step B, the complex isolated by agarose gel filtration selectively forms fd RFII when supplemented with RNase H, RNA polymerase, and the DNA elongation proteins. The omission of any of the proteins described above during the first reaction resulted in either no discrimination or a decrease in discrimination when the missing protein was added during the second step. Results are presented which indicate that E. coli binding protein, discriminatory factors alpha and beta, and RNase H must be present during the time RNA synthesis occurs in order to selectively form RFII from fd DNA and not phiX RFII. The amount of fd and phiX174 RNA-DNA hybrid formed in vitro is directly related to the DNA synthesis observed. Thus, under discriminatory conditions, only fd viral DNA leads to fd RNA-DNA complexes and no phiX RNA-DNA hybrid is formed. Under nondiscriminatory conditions, both DNAs yield RNA-DNA hybrids and DNA synthesis. In the absence of discriminatory factor alpha, no RNA-DNA hybrid is formed with either DNA, and in turn, no DNA synthesis is detected with either DNA template.
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PMID:Selective inhibition of phiX RFII compared with fd RFII DNA synthesis in vitro. II. Resolution of discrimination reaction into multiple steps. 32 48

A simple technique has been developed for isolating intracellular DNA and its bound proteins from uninfected and phage-infected bacteria. This technique, which utilizes aqueous salt concentrations in the physiological range, is based upon the fact that DNA exists in normal cell lysates in a stiff random coil conformation, and has an unusually large excluded volume to mass ratio. Such stiff coils display a unique combination of low sedimentation coefficient and large Stokes radius, enabling them to be separated rapidly from all other cellular components by successive centrifugal and gel permeation steps. Analysis of this purified intracellular DNA fraction from bacteriophage T4-infected Escherichia coli reveals mainly DNA and protein, with a small amount of RNA also present. Among the major proteins obtained are the DNA-dependent RNA polymerase of the host and the products of T4 genes rIIA, rIIB, and 32 (DNA-"unwinding" protein). Small amounts of the proteins coded by T4 genes 43 (DNA polymerase) and 42 (dCMP hydroxymethylase) have also been identified, in addition to at least 13 other phage-coded proteins of unidentified genes. Much of the phage-coded protein in the complex, including the gene 32 protein, does not exchange readily with the same protein exogenously added in the lysate.
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PMID:Intracellular DNA-protein complexes from bacteriophage T4-infected cells isolated by a rapid two-step procedure. Characterization and identification of the protein components. 32 53

Partially purified yeast RNA polymerases (RNA nucleotidyltransferases) initiate DNA synthesis by yeast DNA polymerase (DNA nucleotidyltransferase) I and to a lesser extent yeast DNA polymerase II in the replication of single-stranded DNA. The enzymatic initiation of DNA synthesis on phage fd DNA template occurs with dNTPs alone and is further stimulated by the presence of rNTPs in DNA polymerase I reactions. The presence of rNTPs has no effect on the RNA polymerase initiation of the DNA polymerase II reaction. RNA polymerases I and III are more efficient in initiation of DNA synthesis than RNA polymerase II. Analyses of the products of fd DNA replication show noncovalent linkage between the newly synthesized DNA and the template DNA, and covalent linkage between the newly synthesized RNA and DNA.
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PMID:Enzymatic initiation of DNA synthesis by yeast DNA polymerases. 32 62

Conversion of the viral DNA of phage G4 to the duplex form provided an opportunity to isolate and determine the function of the dnaG protein, the product of a gene known to be essential for replication of the Escherichia coli chromosome. This stage of G4 DNA replication requires action of three proteins: the E. coli DNA-binding protein, the dnaG protein, and the DNA polymerase III holoenzyme. The dnaG protein has been purified approximately 25,000-fold to near-homogeneity. The native protein contains a single polypeptide of 60,000 daltons. It has been assayed for its activity on G4 DNA in three ways: (a) RNA synthesis, (b) complementation for replication of an extract of a temperature-sensitive dnaG mutant, and (c) priming of DNA replication by DNA polymerase III holoenzyme. The dnaG protein is highly specific for G4 DNA and synthesizes a unique 29-residue RNA primer to be described in the suceeding paper. Other single-stranded and duplex DNA templates are inactive. RNA primer synthesis by the dnaG protein has an apparent Km for ribonucleoside triphosphates near 10 micrometer, and a narrow optimum for Mg2+. The sharp specificity of the dnaG protein in choice of template and the utilization of either deoxyribonucleotides or ribonucleotides to produce a hybrid piece only a few residues long (as described in a succeeding paper) suggests that the dnaG protein previously named RNA polymerase by renamed primase.
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PMID:Primase, the dnaG protein of Escherichia coli. An enzyme which starts DNA chains. 34 Apr 57

Adenosine 2',3'-riboepoxide 5'-triphosphate (epoxyATP) has been found to be a suicidal inactivator of DNA polymerase I from Escherichia coli by the following criteria. Inactivation is complete, is first order in enzyme activity, and shows saturation kinetics with an apparent KD of 30 +/- 10 micron for epoxy ATP. This KD is comparable to the KM of the substrate dATP. The t1/2 for inactivation is 1.3 min. Inactivation requires Mg2+ and the complementary template. The enzyme is protected by dATP but not by an excess of template. Gel filtration of the reaction mixture after inactivation with [3H]epoxy ATP results in the comigration of E. coli DNA polymerase I, the tritium-labeled inactivator, and the DNA template. The stoichiometry of binding approaches 1 mol of [3H]epoxy nucleotide per mol of inactivated enzyme. These results are consistent with the hypothesis that epoxy ATP initially serves as a substrate for the polymerase reaction, elongating the DNA chain by a nucleotidyl unit, and subsequently alkylates an essential base at the primer terminus binding site of the enzyme. Epoxy ATP also inactivates human and viral DNA polymerases but not E. coli RNA polymerase or rabbit muscle pyruvate kinase. Hence epoxy ATP may be a specific suicide reagent for DNA polymerases.
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PMID:Apparent suicidal inactivation of DNA polymerase by adenosine 2',3'-riboepoxide 5'-triphosphate. 34 91

In vitro DNA synthesis by yeast DNA polymerase I can be initiated by partially purified yeast RNA polymerases in the presence or absence of rNTPs. Homogeneous yeast RNA polymerase I initiates DNA synthesis by yeast DNA polymerase I on single-stranded DNA templates only in the presence of all four rNTPs. A protein capable of initiating enzymatic DNA synthesis on single-stranded DNA in the absence of rNTPs has also been separated from partially purified yeast RNA polymerase I fractions. Analysis of the RNA polymerase I initiated replication products of phage fd DNA on alkaline sucrose gradients showed noncovalent linkage between the newly synthesized DNA and the template. Isopycnic analyses of the ribonucleotide initiated fd DNA replication products demonstrated covalent linkage between the initiator RNA and newly synthesized DNA. Results from 32P-transfer experiments confirmed the covalent linkage between RNA and DNA chains and showed the presence of all four ribo- and deoxyribonucleotides at the RNA--DNA junctions. The ribonucleotide found most frequently at the RNA--DNA junction is uridylate and the purine deoxynucleotides occur more frequently than pyrimidine deoxynucleotides.
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PMID:Initiation of enzymatic DNA synthesis by yeast RNA polymerase I. 35 89

We report a simple, three-step method for the purification of Escherichia coli DNA polymerase I. Its advantages over other procedures are ease and rapidity, the absence of an autolysis or any high speed centrifugation step, and applicability to large quantities of material. In addition, RNA polymerase can be isolated as a by-product. We have applied this method to purify DNA polymerase both from wild type E. coli cells and from cells bearing a lambda prophage carrying the polA gene (Kelley, W.S., Chalmers, K., and Murray, N.E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5632-5636). This latter source amplifies the amount of DNA polymerase in the cells by at least 10-fold.
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PMID:A simple and rapid purification method for Escherichia coli DNA polymerase I. 38 Dec 83


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