9. Eucaryotic RNA Polymerases

Chambon, Pierre

doi:10.1016/s1874-6047(08)60141-9

Cited by 57 publications

(14 citation statements)

References 182 publications

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“…Rifamycin AF/01-3 which is capable of inhibiting eucaryotic nuclear II and III RNA polymerases (22) gives the same inhibition. a-Amanitin, a potent inhibitor of eucaryotic RNA polymerase type II as well as type III at higher concentrations (8), has no effect on the F. diplosiphon enzymic activity at concentrations up to 10 ,g/ml.…”

Section: Methodsmentioning

confidence: 94%

Purification and Characterization of RNA Polymerase from Fremyella diplosiphon

Miller

Bogorad²

1978

Plant Physiol.

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We have purified and characterized a DNA-dependent RNA polymerase from the blue-green alga Fremyela dipsiphon. This enzyme, purified by gel filtration, DEAE-ceflulose chromatography, and glycerol gradient centrifugation, is comprised of five polypeptide subunits. Their masses are 161,000, 134,000, 91,000, 72,000, and 41,000 daltons. Preparative electrophoresis of the purified enzyme on nondenaturing gels separates the 41,000-dalton polypeptide from the rest of the enzyme. The enzyme is extremely labile in the presence of a variety of salts of strong acids and bases; the purification procedure was devised to avoid exposure to such compounds.The blue-green algae represent a type of procaryotic organization distinct from other bacteria. These photoautotrophs undergo a number of simple developmental processes, such as complementary chromatic adaptation (4), and heterocyst formation (I 1). They also contain Chl a, evolve 02, and bear many structural and biochemical similarities to the chloroplasts of higher plants (7).We have purified and characterized a DNA-dependent RNA polymerase from the blue-green alga Fremyella diplosiphon. The subunit composition of the RNA polymerase from F. diplosiphon reported here is compared with the corresponding enzymes isolated from another blue-green alga, Anacystis nidulans and some bacteria. Determinations. Protein was measured by the Lowry procedure (21), using Cyt c as a standard. DNA concentrations were determined by the diphenylamine method (2) using calf thymus DNA as a standard. Gels were scanned in a Gilford 2000 spectrophotometer equipped with a linear transport assembly (Gilford Instruments Co., Oberlin, Ohio). Standards used to calibrate the Sepharose column and glycerol gradients were detected as follows. Phycoerythrin was assayed spectrophotometrically at 560 nm (3). MATERIALS AND METHODSRibulose bisP carboxylase was assayed using the method of Goldthwaite and Bogorad (10). Catalase was detected by mixing a small aliquot of the fraction to be assayed and a drop of H202; the evolution of 02 bubbles was diagnostic for catalase. ,B-Galactosidase was detected by its A at 280 nm.DNA Isolation. Suspensions of cells of F. diplosiphon or Tolypothrix tenuis disrupted by five cycles of freezing and thawing were diluted with 2 ml/g fresh weight of 1.5 mm Na-citrate, 15 mM NaCl (1/10 SSC; standard saline citrate is 15 mm Na-citrate, 150 mm NaCl) and extracted six times with equal volumes of 1/10 SSC-saturated phenol. The DNA was precipitated from the aqueous phase with 10 volumes of ice-cold absolute ethanol at -20 C. The precipitate was washed once with cold absolute ethanol, twice with cold absolute ethyl ether, and dried under a stream of N2. The DNA was further purified by centrifugation on two CsCl gradients.The first centrifugation was in a block gradient consisting of two layers of CsCl. The bottom layer (2 ml) was adjusted to a density of 1.75 g/cm3 and the top layer (2 ml) to 1.50 g/cm3.

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Section: Methodsmentioning

confidence: 94%

Purification and Characterization of RNA Polymerase from Fremyella diplosiphon

Miller

Bogorad²

1978

Plant Physiol.

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“…Since the kinase can quantitatively convert the 220-kD polypeptide to a 240-kD polypeptide, it may now be possible to test the effect of this phosphorylation in in vitro transcription systems that promote random (reviewed by Chambon, 1974;Roeder, 1976;Lewis and Burgess, 1982) as well as specific initiation of transcription (reviewed by Manley, 1983;Manley et al, 1983). It will also be interesting to test whether phosphorylated RNA polymerase II from plants can substitute for the mammalian enzyme in mammalian transcription systems.…”

Section: Discussionmentioning

confidence: 99%

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

Guilfoyle

1989

Plant Cell

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A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit.

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“…The fo llowing sources arc provided fo r those readers intcrested in some areas of nucleic acid metabolism that are not treated in a comprehensive manner. Unless designated as "recent literature," all are reviews published mainly since 1974: (a) DNA synthesis -DNA synthesis and replication, general (1)(2)(3)(4)(5), Escherichia coli replication (6, 7), bacterial DNA polymerases (8,9), eukaryotic replication and DNA polymerases (10--12), reverse transcriptase (13)(14)(15); (b) trans cription -bacterial transcription (l6- 19), eukaryotic transcription (16,20); (c) recombination-reviews (2 1-23), recent literature (24--26); (d) repair-review (27), recent literature (28)(29)(30)(31)(32); and (e) restriction -modification-reviews (33)(34). Finally, the close re lationship be 56)] belong to the same fa mily as B-DNA but differ from it in having few er residues per turn, a smaller axial rise per residue, a pronounced negative tilt of the base-pair plane, and a helix axis located near or in the minor groove (46).…”

Section: Joyinmentioning

confidence: 99%

Recognition Mechanisms of DNA-Specific Enzymes

Jovin¹

1976

Annu. Rev. Biochem.

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9. Eucaryotic RNA Polymerases

Cited by 57 publications

References 182 publications

Purification and Characterization of RNA Polymerase from Fremyella diplosiphon

Purification and Characterization of RNA Polymerase from Fremyella diplosiphon

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

Recognition Mechanisms of DNA-Specific Enzymes

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