Partial purification and properties of calf thymus deoxyribonucleic acid dependent RNA polymerase III

Weil, P A; Blatti, S. P.

doi:10.1021/bi00679a015

Cited by 61 publications

(47 citation statements)

References 23 publications

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“…Typically, eukaryotic RNA polymerase II is extremely sensitive to ␣-amanitin, whereas RNA polymerase I and RNA-dependent RNA polymerases are not sensitive to this toxin (Lindell et al 1970;Adman et al 1972;White and Wang 1990;Schiebel et al 1993;Goodin et al 1997). ␣-Amanitin does inhibit some RNA polymerase III enzymes, but only at high concentrations (Lindell et al 1970;Weil and Blatti 1975). Synthesis of sense 18S rRNA in P. falciparum nuclei is resistant to ␣-amanitin as previously demonstrated ( Fig.…”

Section: Introductionsupporting

confidence: 77%

RNA polymerase II synthesizes antisense RNA in Plasmodium falciparum

Militello

Patel²,

Chessler³

et al. 2005

RNA

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The recent identification of antisense RNA in the transcriptomes of many eukaryotes has generated enormous interest. The presence of antisense RNA in Plasmodium falciparum, the causative agent of severe malaria, remains controversial. Elucidation of the mechanism of antisense RNA in P. falciparum synthesis is critical in order to demonstrate the origin and function of these transcripts. Therefore, a systematic analysis of antisense and sense RNA synthesis was performed using direct labeling experiments. Nuclear run on experiments with single-stranded DNA probes demonstrated that antisense RNA is synthesized in the nucleus at several genomic loci. Antisense RNA synthesis is sensitive to the potent RNA polymerase II inhibitor ␣-amanitin. Antisense and sense transcription was also detected in nuclei isolated from synchronized parasites, suggesting concurrent synthesis. In summary, our experiments directly demonstrate that antisense RNA synthesis is a common transcriptional phenomenon in P. falciparum, and is catalyzed by RNA polymerase II.

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Section: Introductionsupporting

confidence: 77%

RNA polymerase II synthesizes antisense RNA in Plasmodium falciparum

Militello

Patel²,

Chessler³

et al. 2005

RNA

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“…ld). In animal systems, the RNA polymerase I peak from DEAE-cellulose has been reported to resolve into RNA polymerases I and III on DEAE-Sephadex (2,21,23,24). These two enzymes can be distinguished by the inhibition of RNA polymerase III at 10-3 to 10-4 M a-amanitin while RNA polymerase I is refractory to the toxin (2,21,23).…”

Section: Isolation Of Cauliflower Mosaic Virus Dnamentioning

confidence: 99%

“…4 a to c). RNA (2,21,23,24). RNA polymerase III activity is typically much less than either RNA polymerase I or II activity and has thus proved difficult to detect in a single chromatographic procedure (20,26).…”

Section: Isolation Of Cauliflower Mosaic Virus Dnamentioning

confidence: 99%

“…RNA polymerase II is localized within the nucleoplasm (11, 18), synthesizes heterogeneous and presumably messenger RNA (28), and is inhibited by 10-8 to 10-9 Mi a-amanitin (11,14). RNA polymerase III is localized in the nucleoplasm (18,20) and possibly the cytoplasm (2,20,21,24,26), synthesizes ribosomal 5S and transfer RNAs (25), and is inhibited by 10-3 to 10-4 M a-amanitin in higher animals (2,20,21,24,26), but is refractory to the fungal toxin in lower eukaryotes (1,23,27).Most of the studies on eukaryotic RNA polymerases have been conducted with either animals or lower eukaryotes such as yeast, while studies on higher plants have been limited (3). Plants are known to possess two major forms of RNA polymerase, I and II, and a third form has been described in rye embryos (6).…”

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Purification and Characterization of DNA-dependent RNA Polymerases from Cauliflower Nuclei

Guilfoyle¹

1976

Plant Physiol.

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DNA-dependent RNA polymerases were solubilized from nuclei of cauliflower inflorescences and purified by agarose A-1.5m, DEAEceflulose, DEAE-Sephadex, and Eukaryotes possess multiple forms of DNA-dependent RNA polymerase which transcribe specific DNA cistrons (3, 4, 10).The major forms of eukaryotic nuclear RNA polymerases can be grouped into three species which are referred to as either I or A, II or B, and III or C (3,4,10). RNA polymerase I is localized within the nucleolus (11,13,18), transcribes ribosomal DNA cistrons (9, 17), and is refractory to the fungal toxin, a-amanitin (11,14). RNA polymerase II is localized within the nucleoplasm (11, 18), synthesizes heterogeneous and presumably messenger RNA (28), and is inhibited by 10-8 to 10-9 Mi a-amanitin (11,14). RNA polymerase III is localized in the nucleoplasm (18,20) and possibly the cytoplasm (2,20,21,24,26), synthesizes ribosomal 5S and transfer RNAs (25), and is inhibited by 10-3 to 10-4 M a-amanitin in higher animals (2,20,21,24,26), but is refractory to the fungal toxin in lower eukaryotes (1,23,27).Most of the studies on eukaryotic RNA polymerases have been conducted with either animals or lower eukaryotes such as yeast, while studies on higher plants have been limited (3). Plants are known to possess two major forms of RNA polymerase, I and II, and a third form has been described in rye embryos (6). Here, we describe the separation and purification of multiple forms of nuclear RNA polymerases from cauliflower. Cauliflower inflorescences offer a rich source of RNA polymerases (7,19). The fractionation of three species of RNA polymerase from cauliflower which have properties similar to RNA polymerases I, II, and III described in animals has been achieved in this work.Although the characteristics of the plant enzymes are in general similar to the animal RNA polymerases, a 10-to 100-fold greater concentration of a-amanitin is required to inhibit cauliflower RNA polymerase III than is required to inhibit the animal form III enzyme (20,23

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“…By the use of DEAE-Sephadex chromatography this enzyme, called RNA polymerase C, was detected in other tissues [5,8,9], as well as in cultured cells [3,10,16] and in tumor cells [7], and was identified as the form 111 RNA polymerase first observed by Roeder [I]. RNA poly-[l-101.…”

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confidence: 99%

DNA‐Dependent RNA Polymerase C from Xenopus laevis Ovaries Ability to Transcribe Intact Double‐Stranded DNA

Long

Dina

Crippa

1976

European Journal of Biochemistry

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DN A-dependent RN A polymerase C, partially purified from Xenopus lurvis ovaries, has been resolved by DEAE-Sephadex chromatography in two forms, eluting at 0.2 M and 0.3 M ammonium sulfate, respectively. Both are sensitive to high concentrations of a-amanitin (200 pg/ml). Their ionic strength dependence and divalent cation requirements are indistinguishable. Quantitatively, RNA polymerase C represents the major form of RNA polymerase activity solubilized from the ovaries.Both RNA polymerases C are able to transcribe efficiently either high-molecular-weight Xenopus DNA or intact adenovirus DNA, as compared to nicked DNA. In contrast, RNA polymerase A has little activity on an intact DNA template. The salt dependence of the RNA polymerases C activity is different on the two kinds of template. Nicked DNA is efficiently transcribed up to a salt concentration of 100 mM ammonium sulfate. On intact DNA, optimal transcription is obtained at 40 mM ammonium sulfate and is inhibited by higher salt concentrations.It is now established that three classes of RNA polymerases can be solubilized from eukaryotic tissues RNA polymerase A and RNA polymerase B have been isolated from many different sources and extensively characterized. RNA polymerase A, insentitive to the toxin x-amanitin, is thought to be involved in the transcription of ribosomal RNA, while RNA polymerase B, totally inhibited by 0.1 pg/ml a-amanitin, is probably responsible for the synthesis of heterogeneous nuclear RNA. These two RNA polymerases have been purified to near homogeneity and their subunit composition shown to be different (for a review see [II]). However, RNA polymerase A and B apparently initiate transcription in vitro only at nicks or on denatured regions of the DNA [12-151. The study of their specificity in vitro is thus questionable.A third class of RNA polymerase, sensitive to high concentrations of rx-amanitin, was first observed in cytoplasmic extracts of rat liver [2], and later also in the nuclei of amphibian oocytes [4]. By the use of DEAE-Sephadex chromatography this enzyme, called RNA polymerase C, was detected in other tissues [5,8,9], as well as in cultured cells [3,10,16] and in tumor cells [7], and was identified as the form 111 RNA polymerase first observed by Roeder [I]. RNA poly-[l-101.Enzyme. RNA polymerase or nucleosidetriphosphate: RNA nucleotidyltransferase (EC 2.7.7.6). merase I11 has been shown to be responsible for the synthesis of the 4-S RNA precursor and the 5-S RNA species in nuclei of mouse myeloma cells [17]. After complete purification from plasmacytoma cells, the subunit composition of this enzyme appeared different from the other RNA polymerase classes [18].We report in this paper on the further purification of RNA polymerase C from Xenopus luevis ovaries and on its ability to transcribe intact double-stranded DNA. MATERIALS AND METHODS Chern I culsAmmonium sulfate (ultra-pure), Tris-base (ultrapure) and unlabeled nucleotides were purchased froin Schwarz Mann. Tritiated UTP (ammonium salt, 40 Ci/mmol) was from Ame...

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Partial purification and properties of calf thymus deoxyribonucleic acid dependent RNA polymerase III

Cited by 61 publications

References 23 publications

RNA polymerase II synthesizes antisense RNA in Plasmodium falciparum

RNA polymerase II synthesizes antisense RNA in Plasmodium falciparum

Purification and Characterization of DNA-dependent RNA Polymerases from Cauliflower Nuclei

DNA‐Dependent RNA Polymerase C from Xenopus laevis Ovaries Ability to Transcribe Intact Double‐Stranded DNA

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