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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 more than 2,300-fold purification of a DNA polymerase from the embryos of Drosophila melanogaster is described. The enzyme, which forms a single band on gel electrophoresis, has a molecular weight of about 87,000 and a pH optimum of 8.5. A divalent metal is required for activity, Mg2+ being preferred with activated DNA, Mn2+ with homopolymer template-primers. The enzyme is inactivated completely by mercurials; polyamines are also inhibitory with certain templates. The most efficient template-primer is activated DNA, but homopolymers such as poly(dA)-oligo(dT), poly(A)-oligo(dT), and poly(A)-oligo(U) are also utilized with high efficiency. The purified enzyme preparations appear to be devoid of nuclease activity when assayed directly with suitable substrates. In addition, neither primer nor product is degraded after prolonged incubation with the enzyme. In accordance with previous observations on other DNA polymerases, the Drosophila enzyme can replicate single-stranded DNA only under conditions of simultaneous transcription by RNA polymerase.
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PMID:A DNA polymerase from embryos of Drosophila melanogaster. Purification and properties. 24 52

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

Double-stranded RNA of some virus genomes can be used as template for the DNA-dependent RNA polymerase purified from Escherichia coli. The RNA synthesis requires all four nucleoside triphosphates and manganese ions and is dependent on the presence of sigma subunit. The reaction is inhibited by rifampicin, streptolydigin and ethidium bromide, but not by DNase and actinomycin D which does not bind to double-stranded RNA. The template activity of double-stranded RNA from various viruses is different in each case. The order of template efficiency is Penicillum chrysogenum virus greater than cytoplasmic polyhedrosis virus greater than rice dwarf virus greater than reovirus. The product obtained using cytoplasmic polyhedrosis virus double-stranded RNA as template is single-stranded and hybridizes specifically to the denatured template RNA. One of the major 5'-starting nucleotide sequences of the product RNA is pppA-A-Y--. These results indicate that transcription in vitro of double-stranded RNA by E. Coli RNA polymerase is initiated at specific sites on the template.
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PMID:Transcription of double-stranded RNA by Escherichia coli DNA-dependent RNA polymerase. 32 6

It is shown that the DNA-dependent RNA polymerase of Escherichia coli can synthesize complementary RNA (cRNA) directly on rRNA and mRNA templates. Synthesis occurred preferentially in the presence of Mn2+ and at relatively high substrate and enzyme concentrations. No primer was required, and addition of oligo-U to a mRNA-dependent reaction gave no marked stimulation. Sedimentation analysis of cRNA made on different templates indicated that the products were mainly 2-4 S, but a fraction of the product was larger. Fingerprints of 32P-labelled cRNA made on 5 S rRNA and 18 S rRNA indicated that the complexity of the cRNAs was related to the size of the template, suggesting that a substantial portion of the templates were copied. This reaction provides a simple method for preparing cRNA of high specific activity for use in hybridisation studies, and possibly in sequence analysis. 32P-labelled cRNA made on 18 S and 28 S rRNA was a sensitive hybridisation probe for detection of the specific fragments of mouse DNA containing the rRNA genes.
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PMID:Synthesis of complementary RNA on RNA templates using the DNA-dependent RNA polymerase of Escherichia coli. 33 64

In the presence of Mn2+, globin mRNA can be transcribed into a partial RNA copy by Escherichia coli RNA polymerase. This process also occurs when the mRNA is transcribed together with chromatin. A fraction, at least, of the newly synthesized RNA copy (anti-globin RNA) can serve as a template for the synthesis of globin sequences of the same polarity as the original mRNA. This process is sufficient to explain the specific synthesis of a subset of the globin RNA on mouse foetal liver chromatin. It also accounts for the synthesis of double-stranded RNA sequence by E. coli RNA polymerase, on chromatin as well as on pure mRNA. Results are presented suggesting that the poly(A) tract of the mRNA could be preferentially transcribed. In the presence of Mg2+, the RNA-dependent transcription is strongly inhibited, as well as the synthesis of double-stranded RNA. Under these conditions, the transcription on chromatin appears to be largely DNA dependent, and the synthesis of globin sequences is completely asymmetric. Spermine (0.3 mM) seems to improve the specificity of transcription. The transcription of chromatin in vitro is thus largely dependent on the nature of the divalent cation present in the in the incubation mixture.
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PMID:Specificity of chromatin transcription in vitro. Anomalies due to RNA-dependent RNA synthesis. 37 4

Some properties of unprimed poly(A)-poly(U) synthesis by DNA-dependent RNA polymerase from Caulobacter crescentus were examined. The reaction required ATP and UTP as substrates and manganese as a divalent cation. Rifampicin completely inhibited the reaction at a concentration of 1 micron/ml, and the enzyme catalyzed the polymer synthesis well regardless of the presence of GTP, CTP or both. The chain length of the poly(A)-poly(U) synthesized was about one hundred base pairs, as estimated from a sedimentation velocity and the molar ratio of [3H]AMP to [gamma-32P]ATP incorporated into the poly(A)-poly(U). The reaction was dependent on the square of the enzyme concentration and the enzyme dimers formed complexes with poly(A)-poly(U) during the reaction.
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PMID:Properties of unprimed poly(A)-poly(U) synthesis by Caulobacter crescentus RNA polymerase. 42 57

The transcription of chromatin from adenovrius 2 transformed rat cells by murine plasmacytoma RNA polymerases I, II and III has been studied. Both the total RNA synthesis and transcription of the integrated adenovirus 2 genes by RNA polymerase II represent de novo DNA transcription as assessed by their sensitivity to actinomycin D. It is shown that each RNA polymerase class has characteristic ionic strength activation profiles and metal ion requirements. RNA polymerase II transcribes the integrated adenovirus 2 genes in chromatin at a frequency 25- to 50-fold higher than their sequences are represented in the genome. In contrast, no detectable viral RNA is synthesized when deproteinized DNA is transcribed. In the presence of Mn2+, all three RNA polymerases (I, II and III) transcribe the integrated viral genes at approximately the same relative frequencey. However, the Mg2+ as divalent cation, the proportion of the total RNA which represents viral gene transcripts is increased 3- to 4-fold with RNA polymerase II, while it remains unchanged for RNA polymerases I or III.
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PMID:Transcription of viral genes in chromatin from adenovirus 2 transformed cells by exogenous eukaryotic RNA polymerases. 49 52

The effect of the denaturation of homologous and calf thymus DNA on the RNA polymerase B activity purified from rat liver and spleen and Ehrlich ascites cells, was investigated in presence of either Mn2+ or Mg2+ and in presence or absence of alpha-amanitin. On the basis of the results here reported, we suggest: 1) denatured DNA is more effective than native as template for polymerase B; 2) denatured DNA template and cations might play a role in determining the extent of the reaction alpha-amanitin-polymerase B.
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PMID:[Effect of denaturation of DNA template on activity of RNA polymerase of class B]. 54 76

The effect of low concentrations of actinomycin D was investigated, using two forms of DNA-dependent RNA polymerase (A and B) purified from normal tissues and experimental tumours, in the presence either of Mn2+ or Mg2+, and homologous DNA. The A enzyme activity was strongly inhibited by the antibiotic in presence of Mg2+ and much less in presence of Mn2+. The B enzyme activity was almost suppressed in presence of both cations. The results here reported provide support that the actinomycin D induce a cellular damage of the same extent in normal and tumour tissues.
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PMID:[Effect of actinomycin D on purified DNA-dependent RNA polymerases from normal and neoplastic tissues]. 54 77

A fraction of nucleoli is isolated from zooflagellates (Crithidia oncopelti) nuclei, its DNA-dependent RNA polymerase activity is studied at different temperature, ionic strength and Mg2+, Mn2+ and antibiotic concentrations. The effect of some factors and alpha-amantine on RNA polymerase activity of exonucleolar chromatin was studied as a control. A comparison of heat denaturation of nucleoli and chromatin RNA polymerase activities within the temperature range 30--55 degrees C has revealed a higher thermosensitivity of nucleoli RNA polymerase. Substitution of Mg2+ with equivalent amount of Mn2+ results in a considerable decrease of rRNA synthesis in nucleoli. Nucleoli RNA polymerase activity in the presence of Mg2+ is sensitive to the elevation of ionic strength from 0.12 to 1.30 u; chromatin RNA polymerase activity in the presence of Mn2+ is maximal at high ionic strength (1.30 mu). alpha-Amantine and cycloheximide at high concentrations (10 and 200 mkg/ml) practically do not affect RNA polymerase activity of nucleoli. Nucleoli RNA polymerase of zooflagellates (Crithidia oncopelti) is similar to the A-form of the enzyme in higher eukaryotes.
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PMID:[DNA-dependent RNA polymerase activity of isolated zooflagellates (Crithidia oncopelti) nucleoli]. 56 56


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