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)

Preparations of purified and disrupted suspensions of Coxiella burnetii are able to incorporate ribonucleotides into polymers in the presence of adenosine, guanosine, cytidine, and uridine triphosphates. Nucleotide incorporation requires the presence of all four ribonucleoside triphosphates. The reaction is enhanced by the addition of phosphoenolpyruvate and pyruvic kinase, and exogenous deoxyribonucleic acid, and is inhibited by deoxyribonuclease and actinomycin D. Incorporation is maximal between pH 7.0 and 8.0, and at 37 C. The synthesized polymer is relatively insensitive to deoxyribonuclease and is sensitive to ribonuclease and dilute alkaline hydrolysis. The data indicate the presence of an autonomous deoxyribonucleic acid-dependent ribonucleic acid polymerase in the rickettsial agent.
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PMID:Physiology of rickettsiae. VI. Host-independent synthesis of polyribonucleotides by Coxiella burnetii. 602 13

The mechanism of transcription by Escherichia coli RNA polymerase holoenzyme of chromatin assembled in vitro has been studied by two approaches. Using digestion with endodeoxyribonuclease EcoRI as a probe of mobility, it was found that nucleosome movement is slow compared to the time taken for RNA polymerase to transcribe through regions organised into nucleosomes. However, transcription leads to at least some displacement of nucleosomes relative to their original site on the DNA. In the second approach chromatin was reconstituted from extensively crosslinked histone octamers and simian virus 40 DNA. RNA chain elongation on this template is inhibited relative to non-crosslinked chromatin. This can be related to a decrease in the ability of the cross-linked histone octamers to dissociate from the DNA.
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PMID:Studies on the mechanism of transcription of nucleosomal complexes. 624 43

Linear simian virus 40 DNA has been transcribed in vitro with wheat germ RNA polymerase II. Transcription products have been fractionated on polyacrylamide gels and several discrete sized RNA bands are seen. The RNA band pattern is affected dramatically by deoxyribonuclease treatment during RNA isolation. This is because most of the RNA synthesized is covalently linked to DNA. This linkage has been demonstrated by density analysis in formaldehyde-CsCl gradients and by incorporation of alkali-stable ribonucleotides into DNA. The linear DNA templates transcribed were generated by treatment of circular DNA with restriction enzymes which, in addition to cutting once at a single primary site, were found also to produce single strand nicks at specific secondary sites. The discrete sized RNA bands observed result from initiation at these nicks and terminated at DNA ends. There are two modes of nick-dependent initiation. In one mode the 3'-hydroxyl terminus of the DNA at a single strand nick serves as a primer for the extension of an RNA chain. In a second mode de novo initiation of an RNA chain is promoted at the nick. RNAs which are not primed initiate predominantly with GTP. The catalytic action of wheat germ RNA polymerase II is similar to that of Escherichia coli core RNA polymerase which has also been shown to synthesize primarily RNA which is covalently linked to DNA.
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PMID:Transcription of simian virus 40 DNA by wheat germ RNA polymerase II. Priming of RNA synthesis by the 3'-hydroxyl of DNA at single strand nicks. 624 89

A protein factor which stimulates DNA polymerase alpha activity on heat-denatured DNA has been purified from mouse FM3A cells. The final preparation had a specific activity of 43,000 units/mg protein and lacked detectable DNA polymerase, RNA polymerase, DNA-dependent- and independent ATPase, exo- and endodeoxyribonuclease and phosphatase activities. The stimulating factor sedimented at 2.9S in a glycerol gradient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the glycerol gradient fraction revealed the presence of a major band of 36,000 daltons, the amount of which corresponded well with the level of stimulating activity. The stimulation by the factor was specific for heat-denatured DNA, and a little or no stimulation was observed with native DNA, ribo- and deoxyribohomopolymers and single stranded circular DNA. Alkaline sucrose gradient sedimentation analysis of the reaction products revealed that newly synthesized DNA was covalently linked to the termini of heat-denatured DNA. The average chain length of the elongated span determined by the digestion with micrococcal nuclease and phosphodiesterase II, did not differ between in the presence and absence of the stimulating factor, suggesting that the stimulation by the factor was due to the increase in the initiation frequency of DNA synthesis from the 3'-hydroxyl terminus of heat-denatured DNA.
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PMID:Purification and characterization of a factor stimulating DNA polymerase alpha activity from mouse FM3A cells. 632 2

Essentially all of the DNA polymerase alpha activity in CV-1 monkey cells could be extracted as an enzyme complex that used DNA substrates with a low primer:template ratio, such as denatured DNA, at least 25 times more efficiently than did purified alpha polymerase. This form of the enzyme was rapidly dissociated either by the nonionic detergent Triton X-100 or by chromatography on phosphocellulose to generate alpha polymerase and its protein cofactor complex, C1C2. Both alpha polymerase and C1C2 were then independently purified free of deoxyribonuclease, RNA polymerase, DNA ligase, and ATPase activities, and the C1C2 complex was shown to consist of at least two proteins. Purified C1C2, which exhibited no DNA polymerase activity, completely restored the ability of alpha polymerase to use denatured DNA. Although high concentrations of denatured DNA inhibited the activity of C1C2, which binds tightly to single-stranded but not double-stranded DNA, low concentrations catalyzed reconstitution of alpha polymerase with C1C2. The resulting enzyme complex was chromatographically distinct from alpha polymerase on DEAE-Bio-Gel, was no longer dependent upon addition of C1C2 in order to utilize denatured DNA as effectively as DNase I-activated DNA, and was not inhibited by high concentrations of denatured DNA. These properties of the purified reconstituted enzyme were indistinguishable from those native alpha X C1C2-polymerase.
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PMID:Preparation of DNA polymerase alpha X C1C2 by reconstituting DNA polymerase alpha with its specific stimulatory cofactors, C1C2. 688 71

We have measured the DNA damage formation and repair in the ribosomal and the dihydrofolate reductase (DHFR) genes after treatment of hamster cells with different types of DNA damaging agents. In mammalian cells, the ribosomal DNA (rDNA) is transcribed by RNA polymerase I, whereas the DHFR is transcribed by RNA polymerase II, whereas the DHFR is transcribed by RNA polymerase II. Cells were treated with agents that induce different types of lesions, and that are known to be repaired via different pathways. We used UV (254 nm) irradiation, treatment with cisplatin and treatment with the alkylating agents nitrogen mustard (HN2) and methyl methanesulphonate (MMS). UV induced pyrimidine dimers were detected with the enzyme T4 endonuclease V, which creates nicks at the dimer sites; the breaks are then resolved and identified by denaturing electrophoresis and Southern blot. Intrastrand adducts formed by the alkylating agents HN2 and MMS were quantitated by generating strand breaks at abasic sites after neutral depurination. Interstrand crosslinks (ICL) formed by HN2 and cisplatin were detected by a denaturation-reannealing reaction before neutral agarose gel-electrophoresis. We find that the repair of the pyrimidine dimers is significantly less efficient in the RNA polymerase I transcribed rDNA genes than in RNA polymerase II transcribed DHFR gene at 8 and 24 h after irradiation. ICL and intrastrand adducts induced by HN2 are also removed more slowly from the rDNA than from the DHFR gene. In contrast, MMS induced intrastrand adducts and cisplatin induced ICL are repaired equally efficiently in the RNA polymerase I and RNA polymerase II transcribed genes. We conclude that for some types of DNA damage, there is less repair in the ribosomal genes than in the DHFR; but for other DNA lesions there is no difference. The difference in repair efficiency between the rDNA and the DHFR genes may reflect the different RNA polymerase involved in their transcription. It may, however, alternatively, reflect the different nuclear localization of these genes.
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PMID:Repair of ribosomal RNA genes in hamster cells after UV irradiation, or treatment with cisplatin or alkylating agents. 835 43

Expression of recombinant herpes simplex virus type 1 (HSV-1) deoxyribonuclease (DNase) was analyzed in BHK-21 cells, a standard cell line for virus propagation, by using mammalian cell expression systems based on vaccinia virus and on Semliki Forest virus (SFV)1. Although the establishing of recombinant vaccinia virus failed due to the apparent toxicity of the herpesviral enzyme, soluble and functional HSV-1 DNase was efficiently expressed in BHK-21 cells by the vaccinia virus/T7 RNA polymerase hybrid system as well as by recombinant Semliki Forest virus. Using rabbit antiserum ExoC, directed against the C-terminal residues 503-626, or mouse monoclonal antibody (MAb) Q1, raised against the type 2 enzyme, a major 85-kDa protein with the identical size of the enzyme from HSV-1-infected cells was identified to be induced in both expression systems. With recombinant SFV functional HSV-1 DNase coincided with the overproduction of a single major 85-kDa protein reaching an optimum between 16 h and 36 h after infection. At later times of infection the enzymatic activity vanished. Thus, recombinant SFV may be an appropriate expression vector for biochemical studies of the enzyme when (i) packaged recombinant virus particles are used for infection and (ii) infection does not exceed 24 h. Due to the limitations of transient expression systems, the vaccinia/T7 RNA polymerase hybrid system is suited for expression analysis on a small scale, and for studying intracellular interactions of the enzyme as demonstrated by immunofluorescence microscopy studies. Using vector pTM1, recombinant HSV-1 DNase was efficiently overproduced in BHK-21 cells at 6 h after transfection and was shown to colocalize with the cellular chromatin at sites apparently distinct from the bulk of the herpesviral replication sites the way it is observed for the enzyme of lytically infected cells. The deleting of the 123 C-terminal amino acid residues did not alter this nuclear localization of HSV-1 DNase, suggesting that the latter sequences and other herpesviral factors are not required for the chromatin association.
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PMID:Expression analysis of recombinant herpes simplex virus type 1 DNase. 985 86

After synthesis of short, nascent oligonucleotide in the presence of (32P)DNA, GTP, CTP, UTP and 3'dATP, one can excise with deoxyribonuclease a ternary complex of RNA polymerase, protected DNA and oligonucleotide, while the enzyme simply bound to the template is removed by increasing the ionic strength. This ternary complex is retained on nitrocellulose membranes. On polyacrylamide gel electrophoresis it migrates faster than RNA polymerase alone. The protected portion of the DNA is constituted of about 75 nucleotides. It might represent the sites for RNA initiation.
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PMID:??? 1194 67

Sen1p in Saccharomyces cerevisiae is a Type I DNA/RNA helicase. Mutations in the helicase domain perturb accumulation of diverse RNA classes, and Sen1p has been implicated in 3' end formation of non-coding RNAs. Using a combination of global and candidate-specific two hybrid screens, eight proteins were identified that interact with Sen1p. Interactions with three of the proteins were analyzed further: Rpo21p(Rpb1p), a subunit of RNA polymerase II, Rad2p, a deoxyribonuclease required in DNA repair, and Rnt1p (RNase III), an endoribonuclease required for RNA maturation. For all three interactions, the two-hybrid results were confirmed by co-immunoprecipitation experiments. Genetic tests designed to assess the biological significance of the interactions indicate that Sen1p plays functionally significant roles in transcription and transcription-coupled DNA repair. To investigate the potential role of Sen1p in RNA processing and to assess the functional significance of the Sen1p/Rnt1p interaction, we examined U5 snRNA biogenesis. We provide evidence that Sen1p functions in concert with Rnt1p and the exosome at a late step in 3' end formation of one of the two mature forms of U5 snRNA but not the other. The protein-protein and protein-RNA interactions reported here suggest that the DNA/RNA helicase activity of Sen1p is utilized for several different purposes in multiple gene expression pathways.
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PMID:Multiple protein/protein and protein/RNA interactions suggest roles for yeast DNA/RNA helicase Sen1p in transcription, transcription-coupled DNA repair and RNA processing. 1512 1

Partially purified nuclei from citrus exocortis viroid (CEV)-infected Gynura aurantiaca are able to synthesize linear and circular viroid molecules. Pretreatment of the nuclei with actinomycin D or deoxyribonuclease did not affect viroid synthesis, whereas the synthesis of other cellular RNAs was severely reduced. These observations support the essential role of CEV complementary RNA sequences in viroid replication. However, when alpha-amanitin was included in this in vitro synthesis system, CEV replication was markedly reduced by concentrations of 10 nM or greater. Taken together, these data support the proposition that viroid synthesis is catalyzed by a DNA-dependent RNA polymerase acting on a RNA template.
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PMID:Properties of a cell-free system for synthesis of citrus exocortis viroid. 1659 39


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