Characteristics of DNA‐dependent RNA polymerase activity from isolated yeast nuclei

Brogt, Th.M.; Planta, Rudi J.

doi:10.1016/0014-5793(72)80014-0

Cited by 50 publications

(14 citation statements)

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“…3,1973 on April 27, 2019 by guest http://aac.asm.org/ whether thiolutin also inhibits the processing of rRNA species in yeast. The stable RNA species (15s and 27s) were labeled by growing S. cerevisiae cells in the presence of "4C-adenine, and the cells were converted to spheroplasts.…”

Section: Resultsmentioning

confidence: 99%

“…If thiolutin, DNA template, the other assay components, and enzyme were rapidly mixed in that order at 0 C and subsequently incubated for 20 min at 30 C, the inhibition of RNA synthesis by thiolutin was reduced (A). though three peaks have been reported (3). On the other hand, fractionation on DEAESephadex usually gives a third peak of activity (III) eluting after II.…”

Section: Resultsmentioning

confidence: 99%

“…Thiolutin also inhibits DNA synthe-VOL. 3,1973 on April 27, 2019 by guest http://aac.asm.org/ sis in S. cerevisiae (unpublished data); however, because of difficulties in monitoring DNA synthesis in yeast, these experiments are not conclusive. A direct demonstration of the site of action of thiolutin might come from studies of thiolutin-resistant mutants of S. cerevisiae.…”

Section: Resultsmentioning

confidence: 99%

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Mode of Action of Thiolutin, an Inhibitor of Macromolecular Synthesis in Saccharomyces cerevisiae

Jiménez

Tipper

Davies

1973

Antimicrob Agents Chemother

106

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The sulfur-containing antibiotic thiolutin has been shown to be a potent, reversible inhibitor of the growth of Saccharomyces cerevisiae. Viability was unaffected over the concentration range of 4 to 100 ug/ml. At concentrations as low as 2 sg/ml, the drug inhibited ribonucleic acid (RNA) and protein synthesis in whole cells and spheroplasts. At these low concentrations, protein synthesis continued for a short period of time after RNA synthesis was completely stopped.With higher drug concentrations (greater than 20 ;g/ml) protein synthesis was inhibited; concentrations of thiolutin up to 100 ,ug/ml did not affect translocation or peptide bond formation in cell-free protein-synthesizing systems from yeast.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Mode of Action of Thiolutin, an Inhibitor of Macromolecular Synthesis in Saccharomyces cerevisiae

Jiménez

Tipper

Davies

1973

Antimicrob Agents Chemother

106

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“…Although the original studies of Roeder and of Rutter and coworkers (9) and later of others (10)(11)(12) reported the chromatographic evidence for the presence of polymerase III in yeast, there has never been a confirmation by isolation and characterization of the structure of this enzyme in any lower eukaryotes. In contrast, polymerases I and II have been isolated and their subunit structure defined for yeast (13, 14, t), Dictyostelium (15), and Physarum (16).…”

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confidence: 99%

Molecular structure of yeast RNA polymerase III: demonstration of the tripartite transcriptive system in lower eukaryotes.

Valenzuela¹,

Hager²,

Weinberg³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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Homogeneous RNA polymerase III (RNA nucleotidyltransferase III) has been obtained from yeast. The subunit composition of the enzyme was examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is composed of 12 putative subunits with molecular weights 160,000, 128,000,82,000,41,000, 40,500, 37,000, 34,000,28,000,24,000,20,000,14,500, and 11,000. The high-molecular-weight subunits and several of the smaller subunits of yeast RNA polymerase III are clearly different from those of enzymes I and II, indicating a distinct molecular structure. However, the molecular weights of some of the small subunits (41,000, 28,000, 24,000, and 14,500) appear to be identical to those of polymerases I and II. Thus, it is possible that the three classes of enzymes in yeast have some common subunits.As In lower eukaryotes, however, the situation has remained unclear. Although the original studies of Roeder and of Rutter and coworkers (9) and later of others (10-12) reported the chromatographic evidence for the presence of polymerase III in yeast, there has never been a confirmation by isolation and characterization of the structure of this enzyme in any lower eukaryotes. In contrast, polymerases I and II have been isolated and their subunit structure defined for yeast (13, 14, t), Dictyostelium (15), and Physarum (16).In this paper we report the characterization of yeast polymerase III which can be isolated by two different procedures. The homogeneous preparation contains two large polypeptides (160,000 and 128,000 daltons), one polypeptide of intermediate size (82,000 daltons), and 9 to 10 smaller peptides with stoichiometries varying from 1 to 2 moles per mole of enzyme. This structure is clearly different from yeast polymerases I and II and bears a general resemblance to polymerases III from Xenopus and mouse plasmacytoma. These results provide a convincing argument for the existence of class III polymerases in all eukaryotes and establish a structural basis for the discrimination of the three enzymes.MATERIALS AND METHODS Materials. Materials and yeast cells were obtained as described previously (13). DNA-cellulose was prepared by the method of Alberts and Herrick (17), using denatured calf thymus DNA.RNA Polymerase Assay. Enzymes were assayed as described previously (13). One unit corresponds to the incorporation of 1 nmol of UMP into RNA in 10 min at 300.Enzyme Purification. Yeast RNA polymerase III was purified to homogeneity by two methods. Method A: Polymerase III was obtained as a by-product of a large-scale purification of yeast polymerase I. The first three steps are batchwise treatment with phosphocellulose, batchwise treatment with DEAE-cellulose, and ion-filtration chromatography on DEAE-Sephadex. The details of these procedures, which are common to the purification of polymerase I, will be published elsewhere (13). Enzymes I and III are then separated by DNA-cellulose chromatography (Fig. 1). Whereas, enzyme I is eluted at 0.45 M KCI, polymerase III binds more stron...

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“…The development of methods for the isolation of yeast nuclei in quantities sufficient for enzyme extraction and partial purification has resulted in the localization of at least three nuclear RNA polymerase activities in 5'. ccrrlsbergensis (5) and two DNA polymerase activities in S . cerevisicle (6.…”

Section: Introductionmentioning

confidence: 99%

A DNA endonuclease isolated from yeast nuclear extract

Bryant¹,

Haynes²

1978

Can. J. Biochem.

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We have isolated and partially purified a DNA endonuclease from nuclei of the yeast Saccharomyces cerevisiae. Although purified on the basis of its ability to degrade denatured DNA, the enzyme can also attack native DNA. Denatured oligonucleotide products of the enzyme are sensitive to venom phosphodiesterase (EC3.1.4.1.) but not to bovine spleen phosphodiesterase (EC3.1.4.18). The enzyme has an estimated molecular weight of 6.6--7.5 X 10(4), more than twice as large as the endonucleases involved in DNA repair in Escherichia coli. When analyzed on glycerol gradients, the endonuclease sedimented as a single activity against both denatured DNA and closed circular DNA duplexes. The enzyme showed a 10-fold preference for denatured over native T7 DNA substrate, and appears to produce random nicks in a supercoiled replicative form of phiX174 DNA (RFI) with no discernable preference for the unpaired bases in the supercoiled duplex. The endonuclease appears to be distinct from the yeast endonucleases previously described.

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Characteristics of DNA‐dependent RNA polymerase activity from isolated yeast nuclei

Cited by 50 publications

References 19 publications

Mode of Action of Thiolutin, an Inhibitor of Macromolecular Synthesis in Saccharomyces cerevisiae

Mode of Action of Thiolutin, an Inhibitor of Macromolecular Synthesis in Saccharomyces cerevisiae

Molecular structure of yeast RNA polymerase III: demonstration of the tripartite transcriptive system in lower eukaryotes.

A DNA endonuclease isolated from yeast nuclear extract

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