Purification and Characterization of RNA Polymerase from Fremyella diplosiphon

Abstract

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.

Cyanobacterial RNA polymerase genes rpoC1 and rpoC2 correspond to rpoC of Escherichia coli

“…The rpoCl gene product of N. commune UTEX 584 has an Mr of 71,000, a value which agrees closely with the predicted Mr obtained from sequence analysis (Mr = 70,200). Both of these values are somewhat larger than the values for -y reported in a recent study of 15 taxonomically diverse strains of cyanobacteria (range in Mr, 65,000 to 68,500; 31) and are equivalent to the values reported (Mr = 72,000) for the ry subunits of two other cyanobacteria (10,20).…”

“…The remainder of the derived sequence of RpoC2 and the carboxy-terminal region of the E. coli 1' polypeptide showed only minimal correspondence. A comparison of the sizes of the I' subunits in RNA polymerases from diverse strains of cyanobacteria (10,20,31,32) and that derived from our partial sequence analysis of rpoC2 suggests that approximately 650 codons of rpoC2, with unknown function, remain to be analyzed.…”

“…One question remains puzzling-what is the function(s) of the largest subunit of cyanobacterial RNA polymerase? Mainly because of its size, the largest subunit was considered to be the structural and functional equivalent of the E. coli 3' subunit (10,20,32). As shown here, the derived sequence of N. commiune UTEX 584 rpoCl contained three segments (Fig.…”

Control of Phycoerythrin Synthesis during Chromatic Adaptation

“…At the concentrations used in these experiments rif was a specific inhibitor of RNA polymerase in these cells (11).…”

Specific initiation of transcription at a cyanobacterial promoter with RNA polymerase purified from Calothrix sp. PCC 7601

“…2). A polypeptide of about the same molecuiar mass had been reported by Miller and Bogorad (1978) in the most highly purified preparation of RNA polymerase from the same strain, and was aiso detected when total extract from Catothrix membranacea was probed with the same two antisera that we used (Schneider and Haseikom, 1988a). Schneider and Haselkorn (1988a) concluded that the 90 kDa poiypeptide could represent a product from proteolysis of the p subunit.…”