Purification and Characterization of RNA Polymerase from

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 41,000dalton 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. MATERIALS AND METHODS Cell Culture. Fremyella diplosiphon (B. and F.) Drouet (strain 481) was obtained in axenic culture from the collection at Indiana University (25). The alga was grown in modified medium C (15) with Sequestrene (sodium ferric diethylenetriamine pentaacetate, Geigy Agricultural Chemicals, Ardsley, N.Y.) added to a final concentration of 40 mg/l as a substitute for ferric sulfate. Sodium citrate was omitted entirely. The pH of the medium was adjusted to 7.0 prior to autoclaving. The alga was grown at 37 C in a 14liter Microferm (New Brunswick Scientific Co., New Brunswick, N.J.) equipped with cool-white fluorescent lights. The cells were harvested in late log phase by permitting them to settle to the bottom of the fermenter vessel at 4 C and then concentrating by centrifugation. The packed cells were washed once with 0.05 M TGM2 and stored in liquid N2 until needed. Assay for Polymerase Activity. RNA polymerase activity was 1This work was supported in part by Research Grant PCM 13456 from the National Science Foundation. S. S. M. was a predoctoral trainee of the National Institute of General Medical Sciences during part of this investigation. 2Abbreviation: TGM: 0.05 M Tris-HCI (pH 8.5, 25 C), 4 mM ,-mercaptoethanoL 10% (v/v), glyceroL and 100 Lg/ml phenylmethylsulfonylfluoride. measured as follows: The reaction mixture (1001,l) contained 10 jig of calf thymus DNA (type 1, Sigma Chemical Co.), 8 ,umol of Tris-HCl (pH 8.5), 0.2 gmol each of GTP, UTP, and CTP (Calbiochem), 0.02 ,umol of [14C]ATP (Schwarz/Mann, 53.2 Ci/mol), 3 ,umol of MgCl2, and 0.20 1d of enzyme solution. The mixture was incubated at 36 C for 30 min. The reactions were terminated by addition of 2 ml of ice-cold 5% trichloroacetic acid in 20 mm Na-pyrophosphate. Each precipitate was collected on an 0.45-,um Millipore filter (Millipore Corp.) disc by vacuum filtration, then washed with 20 ml of 5% trichloroacetic acid in 20 mm pyrophosphate and soaked in 10%1o trichloroacetic acid, 20 mm pyrophosphate, 1 M KCI at 25 C for 1 hr. The filters were dried, placed in vials containing Liquifluor (New England Nuclear), and counted in a Packard liquid scintillation spectrophotometer. A unit of activity is defined as 1 nmol of [14CJAMP (1.58 x 103 cpm) incorporated per 30-min assay. 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). Ribulose 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. About 500 Mug ofDNA was loaded onto each gradient and centrifuged at 200,000g for 12 hr in a Beckman Spinco SW 56 rotor. The DNA sediments through the top layer and remains at the interface between the layers. (The contaminating polysaccharides remain at the top and the RNA forms a pellet at the bottom of the tube.) The gradient interface was mixed with 4 ml ofCsCl adjusted to a density of 1.701 g/cm3 and centrifuged in the same rotor at 200,000g for 72 hr. Fractions of 0.2 ml were collected and scanned at 260 nm. The DNA-containing fractions were pooled, dialyzed exhaustively against 1/10 SSC, and stored at -20 C. 995 www.plantphysiol.org on June 24, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved.