Purification Of DNA-dependent RNA Polymerase Research Articles

Effect of heat shock on DNA-dependent RNA polymerase from the cyanobacteriumSynechococcus sp.

Purification of DNA-dependent RNA polymerase from exponentially growing cells of the cyanobacterium Synechococcus sp. is described in cultures grown at normal temperature (39 degrees C) and after heat shock (HS) (47 degrees C). Polyethyleneimine precipitation followed by chromatography and gel filtration steps results in a 39% yield. The enzyme has a component of molar mass of 43 kDa, designated sigma, in addition to the typical procaryotic beta' beta alpha 2 and gamma. The results suggest that Synechococcus RNA polymerase is similar to that of cyanobacterial and E. coli RNA polymerases. Electrophoresis of the HS preparation showed that the enzyme has a component of 18 kDa. This suggests the existence of a functional relationship between this protein and the HS response of Synechococcus RNA polymerase, probably in salvaging denatured RNA polymerase or helping to regain its native structure.

An improved method for the purification of DNA-dependent RNA polymerase from Escherichia coli

DNA-dependent RNA polymerase from Escherichia coli was purified further by elution through heparin-Sepharose CL-6B column after the enzyme was obtained, partially purified, using Burgess and Jendrisak's method [(1975)Biochemistry 14, 4634] The total yield of the pure protein was 10 mg from 50 g of E.coli cells. The method was found to be very reproducible and convenient. The enzyme preparation had 60% active molecules and the elongation rate of RNA synthesis by this enzyme was measured to be 11 bases/s over delta D111 T7 DNA.

Isolation and characterization of DNA-dependent RNA polymerase I of Tetrahymena pyriformis.

A procedure for the separation and purification of DNA-dependent RNA polymerases [EC 2.7.7.6] from macronuclei of Tetrahymena pyriformis is described. We have used it to isolate and characterize the class I enzyme. RNA polymerase I was identified by its resistance against alpha-amanitin and its location in nucleoli. The purified enzyme consists of at least 12 major subunits with approximate molecular weights of 180,000, 118,000, 37,500, 36,000, 29,000, 27,500, 20,000, 18,500, 15,600, 14,500, 13,500, and 12,600. Chromatography on DEAE-Sephadex separated two forms of RNA polymerase I which differed in the presence of an additional polypeptide of 25 kDa. Independently of this polypeptide, the enzyme was found to segregate on DNA cellulose into a binding and a non-binding fraction. This type of heterogeneity was found to be unrelated to differences in molar ratios or molecular weights of the enzyme subunits. The catalytic properties of all enzyme subfractions were very similar and complied with the general characteristics of RNA polymerase I [cf. Roeder, R.G. (1976) in RNA Polymerase (Losick, R. & Chamberlin, M., eds.) pp. 285-329, Cold Spring Harbor Publ. Co., New York].

A form of the DNA-dependent RNA polymerase of Halobacterium halobium, containing an additional component, is able to transcribe native DNA.

A revised procedure for the purification of DNA-dependent RNA polymerase from Halobacterium halobium, including two-phase partition, yields pure, highly active and absolutely DNA-dependent enzyme. Two forms of the enzyme, one containing, the other not containing a previously not observed component, epsilon, show striking differences in activity. RNA polymerase without component epsilon has a significant activity on poly[d(A-T)] but only insignificant activity on all other templates. The enzyme containing a stoichiometric amount of component epsilon transcribes poly[d(A-T)] and native templates efficiently.

Glutamine synthetase in preparations of RNA polymerase of Escherichia coli: a novel purification procedure.

A novel procedure of operational ease and reproducibility for the partial purification of DNA-dependent RNA polymerase [EC 2.7.7.6] from Escherichia coli is reported. It utilizes liquid phase partitions with polyethylene glycol and Dextran, ammonium sulfate fractionations and chromatography on a QAE-Sephadex A-50 column. A copurified protein in the partially purified preparation was isolated and identifed as glutamine synthetase [L-glutamate : ammonia ligase (ADP) EC 6.3.1.2].

Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ

A rapid and simple, large-scale method for the purification of DNA-dependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented. The method involves enzyme extraction at low ionic strength, polyethyleneimine fractionation, (NH(4))(2)SO(4) precipitation, and chromatography on DEAE-Sepharose CL-6B, DEAE-cellulose, and heparin agarose. Milligram quantities of highly purified enzyme can be obtained from kilogram quantities of starting material in 2 to 3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that RNA polymerase III contains 14 subunits with molecular weights of: 150,000; 130,000; 94,000; 55,000; 38,000; 30,000; 28,000; 25,000; 24,500; 20,500; 20,000; 19,500; 17,800; and 17,000. Subunit structure comparison of wheat germ RNA polymerases I, II, and III indicates that all three enzymes may contain common subunits with molecular weights 20,000, 17,800, and 17,000. In addition, RNA polymerases II and III may contain a common subunit with a molecular weight of 25,000, and RNA polymerases I and III may contain a common subunit with a molecular weight of 38,000.

Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I.

RNA Polymerase from the Fungus Aspergillus nidulans

The DNA-dependent RNA polymerase I (or A) from the lower eukaryote Aspergillus nidulans has been purified on a large scale to apparent homogeneity by homogenizing the fungal hyphae in liquid nitrogen, extraction of the enzyme at high salt concentration, precipitation of RNA polymerase activity with polymin P (a polyethylene imine), elution of the RNA polymerase from the polymin P precipitate, ammonium sulphate precipitation, molecular sieving on Bio-Gel A-1.5m, binding to ion-exchangers and DNA-cellulose affinity chromatography. By this procedure 1.6 mg of RNA polymerase I can be purified over 2000-fold from 500 g wet weight of starting material with a yield of 30--35%. The isolated RNA polymerase I is stable for several months at -20 degrees C. The subunit compostion has been resolved by polyacrylamide gel electrophoresis on two-dimensional gels, using either non-denaturing of 8 M urea (pH 8.7) cylindrical gels in the first dimension and sodium dodecyl sulphate slab gels in the second dimension. The putative subunits have molecular weights of 190,000, 135,000, 63,000, 62,000, 43,000, 29,000, (28,000), 16,000 and probably 13,000 and 12,000. Two distinct forms of RNA polymerase I (Ia and Ib) have been resolved by DEAE-Sephadex A-25 chromatography showing ample differences in enzymatic properties and subunit pattern. Additional information is given on RNA polymerase II (or B) which appears to be highly insensitive to alpha-amanitin at concentrations up to 400 micrograms/ml.

Partial purification of DNA-dependent RNA polymerases from small dense nuclei of mouse brain

Partial purification of DNA-dependent RNA polymerases from small dense nuclei of mouse brain

Characterization of a protein factor stimulating RNA synthesis by DNA-dependent RNA polymerase II from plant cell cultures.

During purification of DNA-dependent RNA polymerase II (or B) from cell cultures of parsley a protein fraction was separated by phosphocellulose chromatography which enhanced RNA synthesis in the presence of native homologous DNA. This 'stimulatory factor' was characterized in respect to some effects on the reaction catalyzed by RNA polymerase II. In the presence of the factor the metal ion requirements as well as the ionic strength for optimal RNA synthesis were markedly changed; addition of the factor to RNA polymerase II purified by cellulose chromatography restored those enzyme properties which had apparently changed upon this purification step. The chain length of the RNA product synthesized is favouring the view that the factor acts mainly by stabilizing the elongation step during transcription. The stimulatory factor was further purified by several steps of column chromatography. As derived from the results of gel electrophoresis under denaturing conditions the factor consists of several small polypeptides. Those of Mr = 26000, 25000 and 14000 apparently have counterparts among the smaller subunits of highly purified RNA polymerase II from parsley cells. Another polypeptide of the factor, with Mr = 30000, was only found in those preparations of RNA polymerase II which had not been subjected to phosphocellulose chromatography.

Properties and subunit composition of RNA polymerase II from plant cell cultures

The purification of DNA-dependent RNA polymerase II (EC 2.7.7.6) from plant cell cultures of Petroselinum (parsley) is described. The procedure during which enzyme I is eliminated includes initial precipitation with (NH 4) 2SO 4, an ultracentrifugation step, gel filtration on Sepharose 4B, chromatography on DEAE-cellulose, DNA-agarose and DEAE-Sephadex. The enzyme purified almost to homogeneity exhibits maximal activity with denatured DNA, and is activated preferentially by Mn 2+; α-amanitin acts as a strong inhibitor. Electrophoresis of the enzyme in the presence of dodecylsulphate indicates that it is composed of seven subunits with mol. wts of 200 000, 180 000, 140 000, 43 000, 26 000, 25 000 and 16 000. The results of molecular weight and molar ratio determinations suggest that Petroselinum RNA polymerase II may exist in two active forms differing only in the composition of their high molecular weight subunits.

Chapter 5 DNA-Dependent RNA Polymerases from Yeasts

This chapter focuses on DNA-dependent RNA polymerases from yeasts. Eukaryotic cells contain multiple forms of DNA-dependent RNA polymerases. These enzymes are localized in different compartments. One enzyme is found in the nucleolus and it synthesizes rRNA. Another RNA polymerase can be isolated from the nucleoplasm that synthesizes the bulk of nucleoplasmic RNA, including mRNA. These different RNA polymerases can be clearly distinguished by their chromatographic behavior, their response to the action of α-amanitin, their subunit structure, and their sedimentation value. Other DNA-dependent RNA polymerases are localized in the mitochondria and in the chloroplasts of eukaryotic cells. Mitochondria1 and chloroplast enzymes differ from nuclear enzymes, as they are smaller and are solubilized only by methods different from those used for nuclear enzymes. In the chapter, methods for the purification of DNA-dependent RNA polymerases from yeasts are described. The chapter discusses the separation of RNA polymerases A and B, isolation and purification of RNA polymerase C, and purification of mitochondrial RNA polymerase. Characterization of the RNA polymerases of yeasts is also discussed in the chapter.

Purification of DNA-dependent RNA polymerases A and B from ovaries of Xenopus laevis.

A method was developed for preparing RNA polymerases A and B from ovaries of adult female Xenopus laevis, avoiding sonication procedures at high ionic strength. The technique involves homogenisation in low ionic strength glycerol buffers, precipitation of contaminants with streptomycin sulphate and concentration of enzymes by ammonium sulphate precipitation; this is followed by resuspension of the protein pellets, centrifugation through glycerol density gradients and chromatography on DEAE‐cellulose and phosphocellulose. A and B enzymes, separated at the DEAE‐cellulose step, were purified 90‐fold and 200‐fold respectively by these procedures and were free of ribonuclease and deoxyribonuclease contamination. The two enzymes exhibited properties known to be characteristic of the major eukaryotic RNA polymerases in their ionic strength optima, cation and template requirements and responses to α‐amanitin.

Mammalian Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerases: I. PURIFICATION AND PROPERTIES OF AN α-AMANITIN-SENSITIVE RIBONUCLEIC ACID POLYMERASE AND STIMULATORY FACTORS FROM HELA AND KB CELLS

Abstract A procedure is described for the purification of DNA-dependent RNA polymerase II of the human tissue culture cell lines HeLa and KB. The enzyme has a molecular weight of approximately 500,000 and in sodium dodecyl sulfate polyacrylamide gels invariably shows 3 subunits with molecular weights of 220,000, 140,000, and 35,000. In addition there perhaps is a fourth subunit of about 25,000 daltons or less in mass and a subunit of 170,000 daltons occurring in variable amounts. RNA polymerase II has a strong preference for single-stranded over double-stranded DNA as template and is most active in the presence of 1.5 to 3 mm Mn++ as divalent cation. We also describe the partial purification of two stimulatory factors (SF-A and SF-B) which specifically stimulate the activity of RNA polymerase II in the presence of double-stranded DNA as template.

Purification of DNA-dependent RNA polymerase from Escherichia coli.

Journal Article Purification of DNA-dependent RNA Polymerase from Escherichia coli Get access MARIKO TADA, MARIKO TADA Laboratory of Biochemistry, Aichi Cancer Center Research InstituteChikusa-ku, Nagoya Search for other works by this author on: Oxford Academic PubMed Google Scholar MITSUHIKO TADA MITSUHIKO TADA Laboratory of Biochemistry, Aichi Cancer Center Research InstituteChikusa-ku, Nagoya Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 67, Issue 1, January 1970, Pages 139–141, https://doi.org/10.1093/oxfordjournals.jbchem.a129226 Published: 01 January 1970 Article history Received: 19 April 1969 Published: 01 January 1970

Extraction and purification of DNA-dependent RNA polymerase from rat liver nuclei.

The enzyme RNA polymerase is difficult to extract from most mammalian cells due to its firm association with chromatin. By a combination of intensive ultrasonic treatment and extraction of the nuclei with 0.75 M NaCl at 0° and pH 8.4 for 1.5 hours approximately 80% of the enzyme can be extracted in a soluble, DNA‐dependent form. The extracted enzyme has been purified by DEAE‐cellulose chromatography and centrifugation on sucrose gradients (5–20%, w/v) containing 10% glycerol. A purification of approximately 250‐fold has been achieved in the peak fraction of the gradient. The enzyme requires all four nucleoside triphosphates, mercaptoethanol, a DNA template and either Mg++ or Mn++, the latter being the preferred ion. Enzyme activity can be stimulated by (NH4)2SO4. No direct stimulation of enzyme activity by cortisol phosphate could be observed.

Isolation and purification of DNA-dependent RNA polymerase from nuclei of human placenta (Nucleoside triphosphate: Ribonucleic acid nucleotidyl transferase, E.C.2.7.7.6.)

Isolation and purification of DNA-dependent RNA polymerase from nuclei of human placenta (Nucleoside triphosphate: Ribonucleic acid nucleotidyl transferase, E.C.2.7.7.6.)