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)

Drosophila Kc cells were utilized to prepare nuclear extracts in which promoter-containing DNA templates were efficiently transcribed by RNA polymerase II. A combination of fractionation schemes was used to identify and partially purify seven activities (factors) which affected the transcription of four different genes in vitro. Reconstructing specific transcription required exogenous RNA polymerase II in addition to these factors. Moreover, the high efficiency of transcription characteristic of the crude extract was preserved in reconstruction reactions. The methods used are presented in detail. Functions were assigned to several of the factors. One essential factor appeared to affect initiation and displayed chromatographic properties unlike any other Drosophila transcription factor previously described. Two factors specifically affected RNA chain elongation. Another activity was a DNase inhibitor required to preserve template integrity in the fractionated system. The remaining three factors were not absolutely essential but affected the specific in vitro transcription either qualitatively or quantitatively. A comparison of these transcription factors with other Drosophila and mammalian transcription factors is made.
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PMID:Fractionation of transcription factors for RNA polymerase II from Drosophila Kc cell nuclear extracts. 381 40

A DNase protection technique is described and applied to the interaction of three lac control proteins with supercoiled lac DNA. The technique uses end-labeled oligonucleotide primers to probe specific DNA regions as an alternative to protocols requiring restriction endonuclease cleavage or blotting. Thus DNA may be probed with high resolution in its native state. It is demonstrated that the introduction of supercoiling into DNA accelerates the rate of lac ps promoter binding by RNA polymerase but does not alter the positions at which polymerase, c-AMP-binding protein, or lac repressor bind to lac DNA.
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PMID:Rapid "footprinting" on supercoiled DNA. 388 2

Mov13 mice carry a single Moloney murine leukaemia virus (M-MuLV) proviral copy in the first intron of the alpha 1(I) collagen gene. Virus insertion interferes with the synthesis of stable alpha 1(I) collagen messenger RNA and causes a recessive lethal mutation. The virus insertion has induced changes of the methylation pattern as well as the chromatin conformation in the mutated gene. Specifically, a DNase-hypersensitive site which is associated with active transcription of the wild-type collagen gene is not present in the mutant allele. The block of collagen expression could be caused by virus-induced instability of collagen mRNA or by impaired initiation of transcription. To distinguish between these possibilities, we have compared the activity of the alpha 1(I) collagen gene promoter in cell lines derived from wild-type and Mov13 embryos by nuclear run-on transcription experiments and S1 mapping of nuclear RNA. We show here that initiation of transcription of the mutant gene is reduced 20-100-fold. This indicates that the virus-induced change of chromatin structure in the promoter region of the mutant gene prevents RNA polymerase from binding to its DNA template. Our results are consistent with the notion that the promoter-associated DNase-hypersensitive site is a prerequisite for rather than a consequence of gene activity.
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PMID:Retrovirus insertion inactivates mouse alpha 1(I) collagen gene by blocking initiation of transcription. 396 Jan 20

Synthesis of Rous sarcoma virus RNA was examined in vitro with a new assay for radioactive virus-specific RNA. Nuclei from infected and uninfected cells were incubated with ribonucleoside [alpha-(32)P]triphosphates, Mn(++), Mg(++) and (NH(4))(2)SO(4). Incorporation into total and viral RNA proceeded with similar kinetics for up to 25 min at 37 degrees . About 0.5% of the RNA synthesized by the infected system was scored as virus-specific, compared to 0.03% of the RNA from the uninfected system and 0.005% of the RNA synthesized by monkey kidney cell nuclei. Preincubation with DNase or actinomycin D completely suppressed total and virus-specific RNA synthesis. alpha-Amanitin, a specific inhibitor of eukaryotic RNA polymerase II, completely inhibited virus-specific RNA synthesis, while reducing total RNA synthesis by only 50%. We conclude that tumor virus-specific RNA is synthesized on a DNA template, most probably by the host's RNA polymerase II.
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PMID:In vitro synthesis of Rous sarcoma virus-specific RNA is catalyzed by a DNA-dependent RNA polymerase. 436 1

Poly(A) polymerase activity is induced during vaccinia virus infection of HeLa cells. The enzyme is maximally induced at 3.5 h postinfection. Partial purification frees the preparation of RNase activity and RNA polymerase activity. ATP is the substrate for poly(A) synthesis. A small amount of poly(A) is produced from added adenosine diphosphate due to the production of ATP by an adenylate kinase present in the preparation. The incorporation of ATP into poly(A) is dependent on divalent cations (Mg(2+) or Mn(2+)) and is not inhibited by UTP, CTP, or GTP. Poly(U) stimulates ATP incorporation; poly(A) and poly(C) have little effect on ATP incorporation, and poly(dT) is extremely inhibitory. RNA prepared from HeLa cells and from the partially purified poly(A) polymerase (the enzyme preparation contains endogenous RNA [Brakel and Kates]) stimulates ATP incorporation by poly(A) polymerase which was subjected to DEAE-cellulose chromatography. RNase's, pancreatic and T(1), inhibit the production of poly(A). DNase has little effect. Poly(U) is able to stimulate poly(A) production in the presence of T(1) RNase.
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PMID:Poly(A) polymerase from vaccinia virus-infected cells. I. Partial purification and characterization. 441 6

The effect of histones on accessibility of DNA to DNase in chromatin of thymus nuclei has been studied by selective extraction of either lysine-rich or arginine-rich histones. It was found that all histones block accessibility but that, weight for weight, lysine-rich histones block much more effectively than do arginine-rich histones. We point to the contrast between accessibility of DNA to DNase and of DNA to RNA polymerase, and to what may be the similarity between accessibility to DNase and DNA polymerase.
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PMID:Blocking by histones of accessibility to DNA in chromatin. 450 81

A cytoplasmic, microsomal bound RNA-dependent RNA polymerase has been purified 2500-fold from rabbit reticulocyte lysates. The synthesis of RNA with the purified enzyme is absolutely dependent on the addition of an RNA template. The best template is hemoglobin messenger RNA, while bacteriophage RNA and poly(A,G) are less active, and DNA is completely inactive as a template. With poly(A,G) as a template, only UTP and CTP are incorporated into polynucleotide chains, indicating that the RNA polymerase is an RNA replicase and not a terminal transferase. With messenger RNA as a template, all four ribonucleoside triphosphates are required for maximal activity. The RNA-dependent RNA polymerase reaction is extremely sensitive to low concentrations of heme, rifamycin AF/013, and ribonuclease and resistant to actinomycin D and DNase. The discovery of RNA-directed RNA synthesis in reticulocytes offers an additional site for control of gene expression in mammalian cells and provides a possible mechanism for amplification of the expression of specific genes.
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PMID:Reticulocyte RNA-dependent RNA polymerase. 451 33

The formation of reovirus double-stranded (ds) RNA and of oligo adenylic acid (oligo A) is inhibited by 5 mug of actinomycin D per ml added at the time of viral infection. Viral proteins are synthesized and assembled into dsRNA-deficient particles under these conditions. The addition of cycloheximide to infected cells during the mid-logarithmic phase of viral replication terminates protein and dsRNA synthesis, but allows continued oligo A synthesis for about 1 h. The (3)H-labeled oligo A formed in the presence of cycloheximide is incorporated into particles whose density in CsCl is identical to that of reovirions. Using the large particulate or virus factory-containing cytoplasmic fraction of infected L-cells, we have established an in vitro system for the synthesis of oligo A. The in vitro product migrates slightly faster in sodium dodecyl sulfate acrylamide gels than marker oligo A. Oligo A synthesis in vitro continues for about 1 h, requires, the presence of only one ribonucleoside triphosphate (ATP), is not inhibited by DNase or RNase, but is abruptly terminated by the addition of chymotrypsin to the reaction mixture. Oligo A formed both in vivo and in vitro is released from the factory fraction by chymotrypsin digestion. The enzymes which catalyze the synthesis of oligo A, dsRNA, and single-stranded RNA all exhibit a similar temperature dependence with an optimum of approximately 45 C. These results indicate that oligo A is formed within the core of the nascent virion after the completion of dsRNA synthesis; they suggest that the oligo A polymerase is an alternative activity of the virion-bound transcriptase and that it is regulated by outer capsomere proteins.
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PMID:Shythesis of reovirus oligo adenylic acid in vivo and in vitro. 485 7

Polyhedral cytoplasmic deoxyribovirus virions contain a DNA-dependent RNA polymerase which catalyzes the incorporation of ribonucleotides into an acid-precipitable product. Treatment of virions with sodium deoxycholate and dithiothreitol resulted in the formation of subviral particles which could be separated from virions by rate zonal centrifugation in sucrose gradients. Subviral particles were RNA polymerase-positive and more active per unit mass of protein than virions. In vitro enzyme activity associated with subviral particles required addition of ribonucleotides, Mg(2+), and exogenous denatured DNA template. Optimal enzyme activity occurred over a broad pH (7.2 to 8.8) and Mg(2+) concentration (2 to 10 mumol) range. The specific activity of the RNA polymerase was maximal at 37 C. Addition of DNase or actinomycin D to the reaction mixture reduced the incorporation of [(3)H]UMP into an acid-precipitable product. The product of the reaction was sensitive to degradation by RNase but not to DNase or Pronase. These data suggest that the enzyme copies DNA into RNA.
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PMID:DNA-dependent RNA polymerase activity associated with subviral particles of polyhedral cytoplasmic dexoyribovirus. 485 85

Mesenchymal cells isolated from the papilla of embryonic tooth germs of the mouse were cultured in a complex medium for five to six days. Liquid nitrogen lysates, prepared from these cells, incorporated nucleoside monophosphates into a cold acid-insoluble product. The product was sensitive to RNase and no product was formed if the lysate was pretreated with DNase. The reaction was sensitive to EDTA and, in its presence, optimum activity was obtained with 2 mM MgCl2. On sucrose gradients, the reaction product was distributed between two broad peaks; one centered about 18S and the other above 28S. The RNA polymerase inhibitor alpha-amanitin inhibited approximately 50% of the activity at a concentration of 10 microgram/ml.
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PMID:Transcriptional activity in lysates of cultured mesenchymal cells from embryonic tooth germs. 615 75


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