Purification and properties of the ribonucleic acid-dependent ribonucleic acid polymerase from Halobacterium cutirubrum

1. The RNA-dependent RNA polymerase from Halobacterium cutirubrum was purified to electrophoretic homogeneity. 2. It requires a single-stranded molecule of RNA or polyribonucleotide as template. 3. Nearest-neighbour analyses of the products formed on random poly(A,U) or alternating poly(A-U) templates and base analysis of the product of synthesis directed by wheat-germ RNA prove that the template is copied accurately. 4. The enzyme initiates new chains with purine ribonucleoside triphosphates. 5. Sucrose-density-gradient analysis of the product indicates that it has a size distribution similar to that of the template. 6. Preliminary amino acid analysis of the RNA-dependent polymerase shows that it contains much less serine than either of the subunits of H. cutirubrum DNA-dependent RNA polymerase. 7. The RNA-dependent enzyme is unable to substitute for either subunit of the DNA-dependent polymerase, and both the latter are devoid of RNA-dependent activity.     

Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase II from the mouse plasmacytoma, MOPC 315.

DNA-dependent RNA polymerase II was purified from the mouse plasmacytoma, MOPC 315. Soluble enzyme was obtained from a nucleoplasmic fraction and subjected to chromatography on phosphocellulose, DEAE-cellulose, and DEAE-Sephadex ion exchange resins and was subjected to sedimentation in sucrose density gradients. A chromatographically homogeneous enzyme was obtained which was purified about 25,000-fold relative to whole cell extracts and which had a specific activity (on native DNA) similar to those reported for other purified eukaryotic class II RNA polymerase preparations. Analysis of purified RNA polymerase II by polyacrylamide gel electrophoresis under nondenaturing conditions revealed three protein bands, designated II-O, II-A, and II-B in order of electrophoretic mobility. The subunit compositions of these nondenatured bands were subsequently analyzed by electrophoresis under denaturing conditions. Each enzyme II form contained subunits with molecular weights of 140,000 (II-c), 41,000 (II-d), 30,000 (II-e), 25,000 (II-f), 22,000 (II-g), 20,000 (II-h), and 16,000 (II-i). Molar ratios were unity for all subunits except subunit II-h which had a molar ratio of 2. Each enzyme form was distinguished by its highest molecular weight subunit. II-O contained subunit II-o (molecular weight 240,000), II-A contained subunit II-a (molecular weight 205,000), and II-B contained subunit II-b (molecular weight 170,000). Total molecular weights for II-O, II-A, and II-B were calculated as 554,000, 519,000, and 484,000, respectively. In addition, the number of RNA polymerase II molecules per MOPC 315 tumor cell was calculated to be about 5 times 10-4.     

Partial purification of a ribosomal ribonucleic acid methylase from rat liver nuclei and methylation of undermethylated nuclear ribonucleic acid from regenerating liver of ethionine-treated rat

S-Adenosylmethionine-dependent ribosomal RNA (rRNA) methylase has been purified approx. 90-fold from rat liver nuclei. The partially purified methylase catalyzes the methylation of base and ribose in hypomethylated nuclear rRNA prepared from the regenerating rat liver after treatment with ethionine and adenine. The enzyme has an apparent molecular weight of about 3 x 10(4) and a sedimentation coefficient of 3.0 S. The enzyme is optimally active at pH 9.5 and sensitive to p-chloromercuribenzoate. Thiol-protecting reagents, such as dithiothreitol, are necessary for its activity, and the enzyme requires no divalent cations for its full activity. This enzyme did not efficiently transfer the methyl group to nuclear rRNA from normal rat liver, compared with hypomethylated nuclear rRNA. Methyl groups were mainly incorporated into pre-rRNA larger than 28 S, and the extent of 2'-O-methylation of ribose by this enzyme was greater than that of base methylation in the hypomethylated rRNA. No other nucleic acids, including transfer RNA (tRNA) and microsomal RNA from normal as well as ethionine-treated rat livers, tRNA from Escherichia coli, yeast RNA, and DNA from rat liver and calf thymus, were significantly methylated by this methylase. These results suggest that partially purified rRNA methylase from rat liver nuclei incorporates methyl groups into hypomethylated pre-rRNA from S-adenosylmethionine.     

Two polypeptides associated with the ribonucleic acid polymerase core of Bacillus subtilis during sporulation.

The ribonucleic acid (RNA) polymerase from log-phase and sporulating cells of Bacillus subtilis was analyzed to determine whether any structural changes occurred during sporulation. The elution pattern of RNA polymerase from a deoxyribonucleic acid (DNA)-cellulose column revealed that sporulating cells at stages III and IV contained a new RNA polymerase fraction in addition to the vegetative holoenzyme (alpha2betabeta'sigma). Stage III cells contained the vegetative holoenzyme and a new enzyme with the composition alpha2betabeta'delta1; the molecular weight of delta1 was 28,000. Stage IV cells contained the vegetative holoenzyme, the delta1-containing enzyme, and another enzyme with the composition alpha2betabeta'delta2. The delta2 factor had a molecular weight of around 20,000. The delta-containing enzymes have a higher affinity for the DNA-cellulose column and a higher specific activity on various templates than vegetative holoenzyme. The simultaneous appearance of these enzymes with vegetative holoenzymes in sporulating cells is consistent with the data found previously with DNA-RNA hybridization studies, which showed that sporulating cells contained both vegetative and sporulation messenger RNAs.     

Isolation and characterization of a ribonuclease from human leukemic blood cells specific for ribonucleic acid of ribonucleic acid-deoxyribonucleic acid hybrid molecules.

A ribonuclease that specifically hydrolyzes RNA in RNA. DNA hybrids has been purified more than 100-fold from human acute leukemic white blood cells. The molecular weight of this enzyme has been estimated as 80,000 by glycerol gradient centrifugation. It requires Mg-2plus for activity and is inhibited by N-ethylmaleimide. The optimum activity is observed at pH 8 (37 DEGREES). It is a heat-labile protein, t 1/2 at 50 degrees being 2 min. Among the substrates examined, (A)n X (dT)m, (I)n X (DC)m, and PHIX-174 DNA X RNA were hydrolyzed efficiently. (U)n X (dA)m showed a slight substrate activity, while (c) n X (dG) m and (G)n X (dC)m were not significantly hydrolyzed. The enzyme is an endonuclease and does not require RNA ends in the substrate molecule. It is capable of converting more than 95% of the RNA portions in hybrid substrates into acid-soluble products which are mono- and oligonucleotides terminated in 3'-OH and 5'-phosphate.     

Immunoglobulin M biosynthesis. Production of intermediates and excess of light chain in mouse myeloma MOPC 104E

1. Conditions have been established for the estimation of molecular weights of proteins by analytical gel filtration and sucrose-density-gradient centrifugation in 2.5m-potassium chloride-1m-sodium chloride; Halobacterium cutirubrum polynucleotide phosphorylase, DNA-dependent RNA polymerase and RNA-dependent RNA polymerase have been studied by these methods. 2. The RNA-dependent polymerase has also been studied by density-gradient centrifugation in the absence of salt. 3. All three proteins are of unusually low molecular weight compared with similar enzymes from non-halophilic bacteria.     

Molecular weights of the ribosomal ribonucleic acid of fungi

Summary The molecular weights of the 18s and 25s ribosomal RNA components of fungi from all major classes were determined by electrophoresis in polyacrylamide gels. The molecular weight of the 18s RNA was found to be very similar for all fungi (range 0.71–0.75 million) and about 4–5% larger than the 18s RNA of HeLa cells and soybean. The molecular weight of the 25s RNA ranged between 1.45 million in the Myxomycetes and 1.30–1.31 million in the Ascomycetes and Basidiomycetes. The differences in the 25s RNA molecular weights between various classes of fungi were interpreted as being in agreement with a monophyletic origin of the Chytridiomycetes, Zygomycetes, Ascomycetes and Basidiomycetes, and independent origins for the Myxomycetes and the Oomycetes. The Hyphochytridiomycete examined could not be placed unequivocally in any group on the basis of its 25s RNA. Fungal RNA extracted with a p-aminosalicylate-triisopropylnaphthalene sulfonate-phenol mixture at 40–60°C contained a high molecular weight aggregate of the 18s and 25s ribosomal RNA; this suggested significant base sequence homology between the two ribosomal RNA species in fungi.     

Purification and subunit analysis of wheat-germ ribonucleic acid polymerase II

A procedure is described for the purification of the alpha-amanitin-sensitive DNA-dependent RNA polymerase [EC 2.7.7.6] from wheat germ. Solubilization of the enzyme activity was achieved by sonication of a crude extract in a high-salt buffer. Purification involved precipitation with protamine sulphate and (NH(4))(2)SO(4), chromatography on DEAE-cellulose and phosphocellulose, and sucrose gradient centrifugation. Under denaturing conditions the enzyme dissociated into five polypeptides with molecular weights and molar ratios of 220000 (0.9), 170000 (0.1), 140000 (1.0), 45000 (0.2), and 40000 (0.4). Approx. 1mg of purified RNA polymerase was obtained as a routine from 100g of starting material.     

Ribonucleic acid-dependent ribonucleic acid polymerase in the immune response.

Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 m column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RAN preparation (iotaRNA) as template made from the spleens of immunized mice but very low activity was found with an RNA preparation made from the spleens of normal mice. Incorporation of 3H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iotaRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12-13 S was most active as a template. It was followed by a fraction corresponding to 6-7 S. Sucrose gradient analysis of the 3H-UTP-labeled product was attempted. Some properties of this enzyme are described.     

Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase.

A poliovirus-specific RNA-dependent RNA polymerase was isolated from a cytoplasmic extract of infected HeLa cells and was shown to copurify with a single virus-specific protein. The polymerase was isolated from cells labeled with [35S]-methionine and was fractionated from other soluble cytoplasmic proteins by ammonium sulfate precipitation, phosphocellulose chromatography, gel filtration on Sephacryl S-200, and chromatography on hydroxylapatite. The activity of the enzyme was measured by using either polyadenylic acid or poliovirion RNA as a template in the presence of an oligouridylic acid primer. A single virus-specific protein that had an apparent molecular weight of 63,000 (p63) was found to copurify with this activity. Host-coded proteins were present in reduced molar amounts relative to p63. Noncapsid viral protein 2 (NCVP2) and other viral proteins were clearly separated from p63 by gel filtration on Sephacryl S-200. Polymerase activity coeluted from the column precisely with p63. NCVP2 was totally inactive as an RNA polymerase and did not stimulate the polymerase activity of p63. The purified enzyme sedimented at about 4S on a glycerol gradient and thus appeared to be a monomer of p63. Two-dimensional gel electrophoresis of the polymerase protein indicated that it had an isoelectric point of about 7.5. Thus, the viral polypeptide, p63, as defined by the above physical parameters, is an RNA-dependent RNA polymerase that can copy poliovirion RNA when oligouridylic acid is used as a primer.     

Identification of a viral protein involved in post-translational maturation of the encephalomyocarditis virus capsid precursor.

Translation of encephalomyocarditis virus RNA in a cell-free system from uninfected Krebs ascites cells results in the synthesis of a major polypeptide product with a molecular weight of approximately 112,000. In contrast, when the viral RNA is translated in a cell-free system from virus-infected cells, this polypeptide is absent and the largest polypeptide produced has a molecular weight of about 100,000. This latter polypeptide comigrates on sodium dodecyl sulfate-gels with in vivo virus capsid precursor A, and the two have identical patterns of CNBr-generated peptides. A polypeptide having a molecular weight of 12,500 is also a major translation product in the system from infected cells (but not from uninfected cells). This polypeptide appears to be generated by cleavage of the NH-2-terminal portion of the viral RNA-dependent polypeptides by a proteolytic activity present in the infected cell-free system. This proteolytic activity copurifies with the 23,000-molecular weight viral capsid protein gamma, found in infected cells, through chromatography on DEAE-cellulose and cellulose phosphate. This suggests that gamma is itself a proteolytic enzyme involved in maturation of the viral capsid precursor.     

Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polymerase

1. DNA-dependent RNA polymerase was purified 150-fold from crude extracts of the extreme halophile Halobacterium cutirubrum. 2. The enzyme requires the presence of native DNA and all four nucleoside triphosphates to incorporate (14)C-labelled nucleoside triphosphate into an acid-insoluble ribonuclease-sensitive product. 3. It has an absolute requirement for both Mn(2+) and Mg(2+). 4. The polymerase requires a high salt concentration for stability, but is markedly inhibited by univalent cations. 5. Its molecular weight is very low compared with that of Escherichia coli RNA polymerase.     

Purification and molecular structure of RNA polymerase from influenza virus A/PR8

The RNA-dependent RNA polymerase of influenza virus A/PR/8 was isolated from virus particles by stepwise centrifugation in cesium salts. First, RNP (viral RNA-NP-P proteins) complexes were isolated by glycerol gradient centrifugation of detergent-treated viruses and subsequently NP was dissociated from RNP by cesium chloride gradient centrifugation. The P-RNA (P proteins-viral RNA) complexes were further dissociated into P proteins and viral RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation. The nature of P proteins was further analyzed by glycerol gradient centrifugation and immunoblotting using monospecific antibodies against each P protein. The three P proteins, PB1, PB2, and PA, sedimented altogether as fast as the marker protein with the molecular weight of about 250,000 Da. Upon addition of the template vRNA, the RNA-free P protein complex exhibited the activities of capped RNA cleavage and limited RNA synthesis. When a cell line stably expressing cDNAs for three P proteins and NP protein was examined, the three P proteins were found to be co-precipitated by antibodies against the individual P proteins. These results indicate that the influenza virus RNA-dependent RNA polymerase is a heterocomplex composed of one each of the three P proteins and that the RNA-free RNA polymerase can be isolated in an active form from virus particles. Furthermore, the three P proteins form a complex in the absence of vRNA.     

Molecular hybridization between rat liver deoxyribonucleic acid and complementary ribonucleic acid

RNA (cRNA) was synthesized in vitro on a template of rat liver DNA and its hybridization with rat liver DNA was studied by using the nitrocellulose-filter method. Sonication of the DNA diminished its apparent capacity to hybridize with RNA by about 50%. This is not due to cross-linkage of DNA molecules, because it could be shown that less than 2% of the sonicated DNA was cross-linked. The effect is due instead to the small size of the sonicated DNA molecules. Below a single-stranded molecular weight of 5×10⁵ the DNA showed a progressive loss of capacity to hybridize with decrease in molecular weight. Evidence is presented suggesting that the apparently diminished capacity of the DNA to hybridize is due to loss of hybridized DNA from the membrane filters. When cRNA at concentrations of up to 25μg/ml is annealed with sonicated total DNA, an apparent hybridization saturation value is found at which about 2.5% of the DNA is hybridized with RNA. Increasing the cRNA concentration tenfold brought about the hybridization of a second component of the DNA approximately equal in amount to the first. The renaturation of rat liver DNA was studied by measuring the fall in the extinction at 260nm and two different components of renaturation were observed within the reiterated fraction of DNA. By hybridizing cRNA with different fractions of rat DNA the two components of the hybridization curve are shown to correspond to the two components of the renaturation curve. The conclusion is drawn that at a cRNA concentration of 250μg/ml most of the reiterated fraction of rat liver DNA is hybridized after annealing for 16h under standard conditions (0.30m-sodium chloride–30mm-sodium citrate at 65°C). Even with such a high cRNA concentration little or no hybridization of the slowly renaturing DNA fraction occurs. It is suggested that the most highly reiterated DNA component is poorly transcribed in vitro.     

Partial purification of a ribonucleic acid cAMP-independent protein kinase from embryonic chicken muscle

A cAMP-indepedent protein kinase (P38 kinase) from embryonic chicken muscle with ability to phosphorylate a 38,000 molecular weight polypeptide and to bind to RNAs has been further characterized. An approximately 2000-fold purification of this enzyme was achieved by a combination of affinity and ion-exchange chromatography. Our studies indicate that this protein kinase can not phosphorylate the small subunit of rabbit reticulocyte initiation factor eIF-2 in the presence of its normal endogenous substrate, nor is it activated over a wide range of concentrations of double-stranded RNA. This P38 kinase is, therefore, distinct from the hemin-regulated translational inhibitor of protein synthesis in rabbit reticulocytes and from the interferon-induced protein kinase identified In several systems.     

Ribosomal ribonucleic acid and ribosomal precursor ribonucleic acid in Anacystis nidulans

The RNA of the blue-green alga Anacystis nidulans contains three ribosomal RNA species with molecular weights of 0.56x10(6), 0.9x10(6), and 1.1x10(6) if the RNA is extracted in the absence of Mg(2+). The 0.9x10(6)mol.wt. rRNA is extremely slowly labelled in (32)P-incorporation experiments. This rRNA may be a cleavage product of the 1.1x10(6)mol.wt. rRNA from the ribosomes of cells in certain physiological states (e.g. light-deficiency during growth). The cleavage of the 1.1x10(6)mol.wt. rRNA during the extraction procedure can be prevented by the addition of 10mm-MgCl(2). (32)P-pulse-labelling studies demonstrate the rapid synthesis of two ribosomal precursor RNA species. One precursor RNA migrating slightly slower than the 1.1x10(6)mol.wt. rRNA appears much less stable than the other precursor RNA, which shows the electrophoretic behaviour of the 0.7x10(6)mol.wt. rRNA. Our observations support the close relationship between bacteria and blue-green algae also with respect to rRNA maturation. The conversion of the ribosomal precursor RNA species into 0.56x10(6)- and 1.1x10(6)-mol.wt. rRNA species requires Mg(2+) in the incubation medium.     

The RNA-dependent DNA polymerase of avian sarcoma virus B77. Binding of viral and nonviral ribonucleic acids to the alpha, beta2, and alphabeta forms of the enzyme.

The extent of binding of various RNA species to the three forms of avian sarcoma virus B77 RNA-dependent DNA polymerase was determined using a sensitive nitrocellulose filter binding technique which was capable of detecting binding reactions with association constants as low as 3 X 10(6) liters X mole-1. All three enzyme forms, alphabeta, beta2, and alpha, bound to all single-stranded RNA species that were tested, including nonviral RNAs. 70 S viral RNA exhibited the highest association constant (about 10(11) liters X mole-1), and a population of virus-derived tRNA molecules from which tRNATrp had been removed, the lowest (about 3000 times lower). The affinity for other RNAs was roughly proportional to their size. The affinity of RNAs for the alphabeta enzyme form always exceeded that for the two others by a factor that depended on the particular RNA, never exceeded 6 and was sometimes as low as 1.2. The association constant of the alphabeta enzyme form with viral 70 S RNA was about 15-fold higher than that with viral 35 S RNA. 35 S RNA annealed to tRNATrp had an association constant that was only 2.5 times higher than that of 35 S RNA alone. This finding suggests that the tertiary structure of 70 S RNA plays a significant role in its affinity for B77 DNA polymerase.