Inhibition of DNA-dependent RNA polymerase reaction of Escherichia coll by an antimicrobial antibiotic, streptovaricin

Mizuno, Satoshi; Yamazaki, Hisaji; Nitta, Kazuo; Umezawa, Hamao

doi:10.1016/0005-2787(68)90086-5

Cited by 72 publications

(19 citation statements)

References 16 publications

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“…insensitivity of the soluble enzymes to inhibition by rifamycin SV indicates that these activities are not due to bacterial contamination (15). The isolation of two enzyme activities from DEAEcellulose columns is consistent with recent findings of other investigators studying RNA polymerase from eukaryotic organisms (5, 7), but differs from the reports of Stout and Mans (16) of a single, soluble maize polymerase based on DEAE-cellulose chromatography.…”

Section: Resultssupporting

confidence: 88%

“…The inhibition of RNA polymerase by a-amanitin (14) and rifamycin SV (15) Effect of Mg++ and Mn + on RNA polymerase activity. For these studies and those described in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…RNA polymerase activity was measured as follows: The reaction mixture (0.10 ml) contained 15 yg of corn-nuclear DNA, 8 The presence of all four nucleoside triphosphates was required for the activity of all the enzymes studied here.…”

mentioning

confidence: 99%

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RNA Polymerases of Maize: Nuclear RNA Polymerases

Strain

Mullinix

Bogorad

1971

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

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Two DNA-dependent RNA polymerases of nuclear origin have been purified from leaves of Zea mays. The discovery that the DNA-dependent RNA polymerase of Escherichia coli consists of a core enzyme plus various protein factors that confer specificity in transcription (1, 2) stimulated efforts to purify RNA polymerase from a wide variety of eucaryotes in an attempt to determine whether this enzyme has the potential for controlling transcription of specific types of genes in higher organisms. Although purification of RNA polymerase from eucaryotic organisms has provided few indications of specificity factors (3, 4), investigations with animals (5-7) and with plants (8, 9) have indicated the existence of several forms of RNA polymerase. At least two enzymes have been purified and characterized from calf thymus (7, 10). Enzyme I is believed to be of nucleolar origin and is involved in ribosomal RNA synthesis, while enzyme II has been isolated from the nucleoplasm and presumably is responsible for the synthesis of DNA-like RNA (5). In addition to their chromatographic behavior on DEAE-cellulose and their salt requirements, these two enzymes can be distinguished by their sensitivity to the toxin a-amanitin that is isolated from the basidiomycete Amanita phalloides (11), and their template specificity. Enzyme II is completely inhibited by a-amanitin, whereas enzyme I is not. Enzyme I prefers native DNA, while enzyme II prefers denatured DNA (5).We have studied the properties of RNA polymerases of nuclear origin from corn leaves (Zea mays) as a first step in determining the involvement of these enzymes in the control of transcription. In addition, the relationships among RNA polymerases of DNA-containing organelles is of interest with respect to the nature of the mechanisms controlling the cell as a whole. The study of nuclear RNA polymerases reported here is part of an effort to elucidate the relationship among RNA polymerases of nuclei and chloroplasts (12). MATERIALS AND METHODSPreparation of DNA-Dependent RNA Polymerases. Maize plants (Z. mays, WF9TMS X B 37, Illinois Foundation Seeds, Inc., Champaign, Ill.) were grown in darkness at 280C for 7 days and exposed to light 12 hr before harvest.The leaves were ground in two volumes per unit weight of a solution of 0.25 M Tris(pH 8.0), 0.50 M sucrose, 0.01 MI MgCI2, 0.01 M 2-mercaptoethanol for 15 see at low speed in a Waring Blendor. The homogenate was filtered through several layers of cheesecloth and two layers of Mliracloth (Chicopee Mills, New York), after which it was centrifuged at 100,000 X g for 90 mm in a Type 30 rotor in a Spinco preparative ultracentrifuge. The specific activity of the supernatant was 35 pmol of AMP incorporated per mg of protein.It was brought to 30% of saturation with ammonium sulfate (Schwarz BioResearch, ultrapure). The precipitate (0-30% ammonium sulfate fraction) was collected by centrifugation at 12,000 X g for 20 min, the supernatant was brought to 50% saturation with (NH4)2S0o4, and the resulting precipitate (30-50% ammonium sulf...

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Section: Resultssupporting

confidence: 88%

“…The inhibition of RNA polymerase by a-amanitin (14) and rifamycin SV (15) Effect of Mg++ and Mn + on RNA polymerase activity. For these studies and those described in Fig.…”

Section: Resultsmentioning

confidence: 99%

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RNA Polymerases of Maize: Nuclear RNA Polymerases

Strain

Mullinix

Bogorad

1971

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

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“…Rifamycin [l] , streptovaricin [2] and their derivatives inhibit the function of DNA-dependent RNA-polymerase of prokaryotes and viruses. As shown by Wehrli et al [3] the rifamycin derivative rifampicin forms a stable complex with RNA-polymerase from &'.…”

Section: Introductionmentioning

confidence: 99%

A rifampicin resistent rna‐polymerase from E. coli altered in the β‐subunit

1969

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“…3-5 g (wet weight) of mitochondria in 20 (10,11). Streptovaricin, which has been shown to act as a bacterial RNA polymerase inhibitor (25), has only a slight inhibitory effect on both nuclear and mitochondrial enzymes. In this respect, mitochondrial enzyme differs from the E. coli enzyme.…”

Section: Preparation Of Mitochondriamentioning

confidence: 99%

DNA-Dependent RNA Polymerase from Yeast Mitochondria

Tsai¹,

Michaelis²,

Criddle³

1971

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

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The DNA-dependent RNA polymerase present in the mitochondria of Saccharomyces cerevisiae has been solubilized using a 0.5 M KCI solution, and the soluble enzyme has been purified. Two forms of mitochondrial enzyme were obtained; they differ in their template specificity and metal-ion dependency. The mitochondrial RNA polymerase also differs from enzyme obtained from the nucleus with respect to antibiotic sensitivity and template specificity. Only the nuclear enzyme is sensitive to a-amanitin inhibition. It is becoming increasingly evident that mitochondria possess a considerable degree of autonomy in their biogenesis (1, 2). That mitochondria can also synthesize their own RNA has been demonstrated in several organisms (3-7). Saccone et al. (8) established that this synthesis is catalyzed by a DNAdependent RNA polymerase localized in the mitochondria and cannot be attributed to contamination by the nuclear enzyme. In this regard, it would be interesting to establish whether the mitochondrial DNA-dependent RNA polymerase differs in chemical and physical properties from the nuclear enzyme. Shmerling (9) demonstrated that rat-liver mitochondrial RNA synthesis differs from nuclear RNA synthesis with respect to rifamycin sensitivity. However, work in our laboratory (10), and studies by Wintersberger and Wintersberger (11), and by Herzfeld (12) were not able to reproduce these results with mitochondria from yeast and Neurospora, respectively. These experiments were performed using intact mitochondria or mitochondrial fragments. In order to make a more careful comparison between the mitochondrial and nuclear DNA-dependent RNA polymerases, it is necessary to isolate and purify these enzymes to a reasonable degree.Tewari and Wildman (13) studied tobacco-chloroplast RNA polymerase, and found it was associated with the chloroplast membrane. They were unable to solubilize this enzyme. Wintersberger and Wintersberger (11) also reported that polymerase was tightly bound to the membrane and that severe disruption of membrane causes loss of enzyme activity. Saccone and Gadaleta (14) have recently reported solubilization of the rat-liver mitochondrial enzyme using the detergent, deoxycholate. 473 We have used 0.5 M KCI to solubilize the enzyme from yeast mitochondrial membrane. After DNase treatment, ammonium sulfate fractionation, and DEAE-Sephadex column chromatography, the enzyme was purified 3400-to 4200-fold. The enzyme purified in this way differs from the whole-cell preparation in its insensitivity to a-amanitin and its template specificity.Nagley and Linnane (15), and Goldring et al. (16) have obtained cytoplasmic petite mutants, by treating yeast with ethidium bromide, that do not have a detectable amount of mitochondrial DNA. We have also prepared a petite mutant lacking detectable mitochondrial DNA by treating yeast with acriflavin. We compared this petite with one in which mitochondrial DNA is still present and only slightly altered in base composition. The petite mutant in which mitochondrial DNA is undet...

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Inhibition of DNA-dependent RNA polymerase reaction of Escherichia coll by an antimicrobial antibiotic, streptovaricin

Cited by 72 publications

References 16 publications

RNA Polymerases of Maize: Nuclear RNA Polymerases

RNA Polymerases of Maize: Nuclear RNA Polymerases

A rifampicin resistent rna‐polymerase from E. coli altered in the β‐subunit

DNA-Dependent RNA Polymerase from Yeast Mitochondria

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