DNA-dependent RNA polymerase I from human placental nuclei

This paper describes a large-scale method for solubilisation and purification of DNA-dependent RNA-Polymerase I from mature human placenta. The solubilisation method involves homogenization of the whole human placenta, isolation of cell nuclei, sonication of separated nuclei at high ionic strength and ammonium sulfate precipitation. The purification method consists of chromatography of RNA-Polymerase I activity on DEAE-Sephadex A-25 and Phosphocellulose P-11, and glycerol-density gradient centrifugation. In result, RNA-Polymerase I of human placenta nuclei has been shown to be completely resistant to alpha-amanitin. Besides dependence of RNA-Polymerase I on different Mg2+ and Mn2+ concentrations, glycerol concentration and ionic strength was studied. Using our results, an optimal RNA-Polymerase I assay mixture was developed. The subunit composition of RNA-Polymerase I was investigated by dodecylsulfate-gel electrophoresis. The RNA-Polymerase I molecule of human placenta consists of 13-14 polypeptides.     

Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase.

An improved purification procedure is described for the sigma subunit of escherichia coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70, and finally Ultragel AcA44. The sigma factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical--physical properties of sigma are presented. A molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of sigma have been determined. The isoelectric focusing properties of sigma are presented. Denaturation--renaturation studies indicate that sigma is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000-dalton fragment is generated from sigma by mild trypsin treatment.     

Large-scale purification and subunit structure of DNA-dependent RNA polymerase II from cauliflower inflorescence

DNA-dependent RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from cauliflower inflorescence (Brassica oleracae, var. botrytis) was highly purified by polyethyleneimine treatment on a large scale. The solubilized enzyme was partially purified by polyethyleneimine fractionation and subjected to chromatography on DEAE-Sephadex and phosphocellulose, and subsequently to sedimentation in a glycerol gradient. The specific activity (231 nmol/mg per 10 min) of this enzyme was comparable to that reported for other purified eukaryotic RNA polymerases. Analysis of the purified RNA polymerase II by polyacrylamide gel electrophoresis under non-denaturing conditions revealed a single band. The subunit composition of the enzyme was analyzed by electrophoresis under denaturing conditions. The RNA polymerase II contained subunits with molecular weights and molar ratios (in parentheses) of 180 000(1), 130 000(2), 48 000(2), 25 000(4), and 19 500(4).     

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].     

Crystal structure of a DNA-dependent RNA polymerase (DNA primase)

Primases are essential components of the DNA replication apparatus in every organism. They catalyze the synthesis of oligoribonucleotides on single-stranded DNA, which subsequently serve as primers for the replicative DNA polymerases. In contrast to bacterial primases, the archaeal enzymes are closely related to their eukaryotic counterparts. We have solved the crystal structure of the catalytic primase subunit from the hyperthermophilic archaeon Pyrococcus furiosus at 2.3 A resolution by multiwavelength anomalous dispersion methods. The structure shows a two-domain arrangement with a novel zinc knuckle motif located in the primase (prim) domain. In this first structure of a complete protein of the archaeal/eukaryotic primase family, the arrangement of the catalytically active residues resembles the active sites of various DNA polymerases that are unrelated in fold.     

DNA-dependent RNA polymerase of thermoacidophilic archaebacteria

Among 979 non-glycerol growers of the yeast Schizosaccharomyces pombe, 40 strains were found to be deficient in the mitochondrial ATPase activity. Three of them exhibited an alteration in either the alpha or beta subunits of the F1ATPase. The alpha subunit was not immunodetected in the A23/13 mutant. The beta subunit was not immuno-detected in the B59/1 mutant. The existence of these two mutants shows that the alpha and beta subunits can be present independently of each other in the inner mitochondrial membrane. The beta subunit of the mutant F25/28 had a slower electrophoretic mobility than that of the wild-type beta subunit. This phenotype indicates abnormal processing or specific modification of the beta subunit. All mutants showed reduced activities of the NADH-cytochrome c reductase and of the cytochrome oxidase and a decreased synthesis of cytochrome aa3 and cytochrome b. This pleiotropic phenotype appears to result from specific modifications in the mitochondrial protein synthesis. The mitochondrial synthesis of four polypeptides (three cytochrome oxidase and one cytochrome b subunits) was markedly decreased or absent while three new polypeptides (Mr = 54000, 20000 and 15000) were detected in all the mutants analysed. This observation suggests that a functional F1ATPase is necessary for the correct synthesis and/or assembly of the mitochondrially made components of the cytochrome oxidase and cytochrome b complexes.     

Purification and characterization of the DNA-dependent RNA polymerase and its subunit sigma from Micrococcus luteus

DNA-dependent RNA polymerase from Micrococcus luteus can be isolated from cell extracts after removal of an excess of nucleic acids by fractionation with ammonium sulfate, followed by two consecutive gel filtrations through agarose and chromatography on cellulose phospate. Either homogeneous holoenzyme or a mixture of core and holoenzyme is obtained in this way, as is indicated by electrophoresis in polyacrylamide gels in the absence of detergent, where core enzyme migrates ahead of holoenzyme. Homogeneous core enzyme can be isolated from holoenzyme by chromatography on DEAE-cellulose. Core enzyme contains the subunits alpha, beta and beta' previously described [U.I. Lill et al., (1975) Eur. J. Biochem. 52, 411-420] in a molar ratio of 2:1:1. Holoenzyme contains an additional subunit sigma of 80 000 molecular weight (molar subunit composition alpha2 betabeta' sigma) and two relatively small polypeptides (molecular weight 14 000 and 25 000, respectively). Subunit sigma may be isolated from holoenzyme by chromatography on DEAE-cellulose at pH 6.9 in the presence of low concentrations of glycerol. The behaviour of holoenzyme during sedimentation in a glycerol gradient at low ionic strength indicates its occurrence as a dimer of the alpha2betabeta'sigma-protomer, whereas the monomeric form is preferred by core enzyme. Holoenzyme is much more active than core enzyme in RNA synthesis on bacteriophage T4DNA as template. The activity of the latter is stimulated by isolated sigma. M. luteus sigma as well as holoenzyme enhances also the activity of core enzyme fro- Escherichia coli. The formation of a hybrid between micrococcal sigma and E. coli core polymerase is also suggested by the influence of sigma on the oligomerisation of the enzyme from E. coli.     

Zinc is associated with the beta subunit of DNA-dependent RNA polymerase of Bacillus subtilis.

The Bacillus subtilis DNA-dependent RNA polymerase holoenzyme and core enzyme each contain approximately two atoms of zinc per molecule. When the dissociated subunits of the enzyme are passed through a blue dextran-Sepharose affinity column, only the beta subunit binds to the column. The total zinc content of the enzyme is tightly bound to the beta subunit. Dialysis studies suggest that the two zinc ions differ in the strength of their association with the beta subunit. The presence of zinc in beta is consistent with several other lines of evidence which indicate that this subunit is dirrectly involved in phosphodiester bond formation. The blue dextran-Sepharose column procedure should be useful in future studies of the dissociation and reassociation of the enzyme since the method is rapid and provides excellent recovery of the beta subunit as well as the alpha and beta' subunits of the RNA polymerase.     

RPA190, the gene coding for the largest subunit of yeast RNA polymerase A

Yeast RNA polymerases are being extensively studied at the gene level. The entire gene encoding the largest subunit of RNA polymerase A, A190, was isolated and characterized in detail. Southern hybridization and gene disruption experiments showed that the RPA190 gene is unique in the haploid yeast genome and essential for cell viability. Nuclease S1 mapping was used to identify mRNA 5' and 3' termini. RPA190 encodes a polypeptide chain of 186,270 daltons in a large uninterrupted reading frame. A dot matrix comparison of the deduced amino acid sequence of subunit A190 with Escherichia coli beta' and cognate subunits B220 and C160 from yeast RNA polymerases B and C showed a conserved pattern of homology regions (I-VI). A potential DNA-binding site (zinc-binding motif) is conserved in the N-terminal region I. Remarkably, the A190 subunit does not harbor the heptapeptide repeated sequence present in the B220 subunit. The sequence of the A190 subunit diverges from B220 and C160 by the presence of two hydrophilic domains inserted between homology regions I and II, and V and VI. From their codon usage and third base pyrimidine bias, RNA polymerase genes RPA190, RPB220, RPC160, and RPC40 fall among yeast genes expressed at an average level. The RPA190 5'-flanking region contains features present in other polymerase genes that might function in regulation.     

RNA polymerase from the fungus, Aspergillus nidulans. Large-scale purification of DNA-dependent RNA polymerase I (or A).

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.