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

1. Crude extracts of the extreme halophile Halobacterium cutirubrum contain separable DNA-dependent and RNA-dependent RNA polymerases. 2. The RNA-dependent enzyme has been purified about 2800-fold. 3. It requires RNA, preferably of high molecular weight, and all four ribonucleoside triphosphates to incorporate (14)C-labelled nucleoside triphosphate into an acid-insoluble, ribonuclease-sensitive product. 4. Both the stability and activity of the RNA polymerase are relatively insensitive to changes in potassium chloride or sodium chloride concentration, but incorporation is stimulated by both Mg(2+) and Mn(2+). 5. The molecular weight of the enzyme is about 17000-18000.     

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.     

Kinetics and template-dependency of ribonucleic acid synthesis by bacterial ribonucleic acid polymerase.

The rate of RNA synthesis catalysed by DNA-dependent RNA polymerase shows a Michealis-Menten-type saturation curve with increasing template concentration. However, the apparent Km is proportional to enzyme concentration, indicating that the reaction does not obey a simple kinetic scheme. The action of inhibitors also indicates a more complex interaction between the enzyme and the DNA template; many inhibitors of RNA synthesis either decrease Vmax. without affecting Km, or increase Km without affecting Vmax. All of these observations can be accounted for quantitatively by a reaction pathway in which the non-specific binding sites of the viral DNA template inhibit competitively the binding of the enzyme to the initiation sites. In terms of this pathway the two classes of inhibitors of RNA synthesis must then act predominantly either on the rate of elongation or on the availability of the binding sites respectively.     

The role of Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polymerase subunits in initiation and polymerization

1. The two subunits alpha and beta of Halobacterium cutirubrum DNA-dependent RNA polymerase are required in equimolar amounts for RNA synthesis to occur in vitro at the maximum rate. 2. In the absence of bivalent cations no interaction occurs between alpha and beta subunits or between the subunits and DNA. 3. Mn(2+) causes the subunits to form a 1:1 complex that still does not bind to the template. 4. Mg(2+) permits binding of the Mn(2+)-mediated complex to DNA. 5. The complete enzyme, alphabeta, is inhibited by rifampicin and only the beta subunit relieves the inhibition when added in excess. 6. Rifampicin-insensitive, template-dependent RNA synthesis occurs in the presence of protein alpha alone provided an oligonucleotide with a 5'-purine terminus is supplied as primer. 7. In the primed reaction with the alpha protein and an oligonucleotide, the template specificity is independent of the ionic strength, in contrast with the marked effect of salt concentration on the template specificity of the complete enzyme. 8. It is concluded that the beta protein controls the specificity of chain initiation and the template specificity of the complete enzyme and also carries the rifampicin-binding site, whereas the catalytic site is on the alpha subunit.     

Replication of RNA by the DNA-dependent RNA polymerase of phage T7

The DNA-dependent RNA polymerase of bacteriophage T7 utilizes a specific RNA as a template and replicates it efficiently and accurately. The RNA product (X RNA), approximately 70 nucleotides long, is initiated with either pppC or pppG and contains an AU-tich sequence. Replication of X RNA involves synthesis of complementary strands. Both strands are also significantly self-complementary, producing RNA with an extensive hairpin secondary structure. Replication of X RNA by T7 RNA polymerase is both template and enzyme specific. No other RNA serves as template for replication; neither do other polymerases, including the closely related T3 RNA polymerase, replicate X RNA. The T7 RNA polymerase-X RNA system provides an interesting model for studying replication of RNA by DNA-dependent RNA polymerases. Such a mechanism has been proposed to propagate viroids and hepatitis delta, pathogenic RNAs whose replication seems to depend on cellular RNA polymerases.     

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.     

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.     

Characterization of DNA polymerase and RNA associated with A-type particles from murine myeloma cells.

The RNA-dependent DNA polymerase present in intracisternal A-type particles from mouse myeloma tumor cells has been studied. This polymerase can use either endogenous A particle RNA or an exogenous synthetic polynucleotide [poly (rA)] as a template. The DNA reaction product is small (4S-10S) and over 90% of it hybridizes to A particle RNA, whereas up to 50% of it hybridizes to murine sarcoma-leukemia virus RNAs. The RNA isolated from purified A particles is generally of low molecular weight (5S-15S) but contains small amount of 70S and 35S components. These results suggest that A-type particles may be related to C-type oncornaviruses.     

The relationship between the deoxyribonucleic acid-bound and low-molecular-weight soluble forms of Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polymerase.

1. Slow, spontaneous lysis of Halobacterium cutirubrum in 3 M-KCl yields DNA-dependent RNA polymerase as a complex with DNA that sediments completely at 45 000g. 2. Controlled deoxyribonuclease digestion of the complex, with or without subsequent sonication, releases the enzyme quantitatively in a soluble form that passes through ultrafilters with a molecular-weight exclusion limit of 50 000. 3. Purification of the active ultrafiltrate by gel filtration and hydroxyapatite chromatography gives a high yield of the purified alpha and beta subunits. 4. The low mol.wt. (17 800-19 000) of the soluble enzyme was confirmed by gel filtration and is unchanged by sonication of the DNA-enzyme complex. 5. A new assay applicable to both forms of the enzyme was developed. 6. The bivalent-cation requirement of the soluble form depends on the buffer concentration. 7. Both the DNA-enzyme complex and the low-molecular-weight soluble forms of the polymerase catalyse formation of short RNA chains only.     

Polyadenylic acid synthesis activity of purified DNA-dependent RNA polymerase from Caulobacter

Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor.     

Isolation and properties of highly purified Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polumerase

1. The subunits α and β of Halobacterium cutirubrum DNA-dependent RNA polymerase have been purified to electrophoretic homogeneity. Both have mol.wt. 18000 and they are required in equimolar amounts for optimum activity. 2. The instability of the complete enzyme, αβ, in the absence of salt is due to the rapid inactivation of the β subunit in these conditions. 3. Nearest-neighbour analysis of the product formed on poly[d(A-T)] as template shows that the enzyme copies the latter accurately. 4. The enzyme initiates new chains with purine nucleoside triphosphates exclusively. 5. The product obtained in the standard assay conditions contains some high mol.wt. (>16S) material, but consists primarily of short chains, of average length 70–80 nucleotide units. 6. The template specificity of the complete enzyme has been studied at high and low ionic strength. Its extreme dependence on salt concentration is unrelated to the gross overall base composition of the DNA used. 7. T₇ DNA is transcribed asymmetrically and the enzyme selectively copies the T₇ `early' genes. 8. Preliminary amino acid analyses of α and β subunits show that their overall content of acidic, basic and neutral amino acids does not differ appreciably from that of Escherichia coli RNA polymerase.     

Inhibition of yeast ribonucleic acid polymerases by thiolutin

Yeast ribonucleic acid (RNA) polymerase II, isolated after fractionation on diethylaminoethyl (DEAE)-cellulose (DE-52) or on DEAE-Sephadex (A-25), is 50% inhibited by 1.5 mug of alpha-amanitin. This inhibition is independent of the sequence of interaction of enzyme, template, nucleotides, and antibiotic and is expressed immediately on addition of alpha-amanitin to a preparation actively synthesizing RNA. Thus, alpha-amanitin's primary effect is inhibition of elongation of preinitiated RNA sequences in this system, as in others. A single peak of alpha-amanitin-resistant RNA polymerase activity (I) was eluted before enzyme II on either column. On A-25 but not on DE-52, a third peak of activity (III) was eluted after enzyme II. This activity was also resistant to alpha-amanitin. Enzymes I, II, and III were 50% inhibited by 3, 4, and 3 mug of thiolutin per ml, respectively. The extent of inhibition was independent of the nature of the template (native or denatured salmon sperm deoxyribonucleic acid or poly(dA-dT) or of the presence of 0.4 mM dithiothreitol, but this marked inhibition was only seen when enzymes were preincubated with thiolutin in the absence of template. Template protected the enzymes against thiolutin in the absence of nucleotides. Either the sensitive site on the polymerase is only accessible to thiolutin before interaction with template or thiolutin inhibits functional polymerase-template interaction but not elongation of preinitiated RNA chains.     

RNA-dependent RNA polymerase activity associated with endogenous double-stranded RNA in rice

RNA-dependent RNA polymerase (RdRp) activity was detected in the crude microsomal fraction of rice cultured cells that contain a 14 kbp double-stranded RNA (dsRNA). RdRp activity is maximal in the presence of all four nucleotide triphosphates and Mg2+ ion and is resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D and alpha-amanitin). RdRp activity increases approximately 2.5-fold in the presence of 0.5% deoxycholate. Treatment of purified microsomal fraction with proteinase K plus deoxycholate suggests that the RdRp enzyme complex with its own 14 kb RNA template is located in vesicles. The RdRp enzyme complex was solubilized with Nonidet P-40 and purified by glycerol gradient centrifugation, then exogenous RNA templates were added. Results indicate that exogenous dsRNA reduces RNA synthesis from the endogenous 14 kb RNA template.     

Mechanism of ribonucleic acid chain initiation. 1. A non-steady-state study of ribonucleic acid synthesis without enzyme turnover

A non-steady-state kinetic method has been developed to observe the initiation of long RNA chains by Escherichia coli RNA polymerase without the enzyme turnover. This method was used to determine the order of binding of the first two nucleotides to the enzyme in RNA synthesis with the first two nucleotides to the enzyme in RNA synthesis with poly(dA-dT) as the template. It was shown that initiator [ATP, uridyly(3'-5')adenosine, or adenyly(3'-5')uridylyl-(3'-5')adenosine] binds first to the enzyme-template complex, followed by UTP binding. The concentration dependence of UTP incorporation into the initiation complex suggests that more than one UTP molecule may bind to the enzyme-DNA complex during the initiation process. Comparison of the kinetic parameters derived from these studies with those obtained under steady-state conditions indicates that the steps involving binding of initiator or UTP during initiation cannot be rate limiting in the poly(dA-dT)-directed RNA synthesis. The non-steady-state technique also provides a method for active-site titration of RNA polymerase. The results show that only 36 +/- 9% of the enzyme molecules are active in a RNA polymerase preparation of high purity and specific activity. In addition, the minimal length of poly(dA-dT) involved in RNA synthesis by one RNA polymerase molecule was estimated to be approximately 500 base pairs.     

Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro.

A soluble RNA-dependent RNA polymerase was isolated from poliovirus-infected HeLa cells and was shown to copy poliovirus RNA in vitro. The enzyme was purified from a 200,000-X-g supernatant of a cytoplasmic extract of infected cells. The activity of the enzyme was measured throughout the purification by using a polyadenylic acid template and oligouridylic acid primer. The enzyme was partially purified by ammonium sulfate precipitation, glycerol gradient centrifugation, and phosphocellulose chromatography. The polymerase precipitated in a 35% saturated solution of ammonium sulfate, sedimented at about 7S on a glycerol gradient, and eluted from phosphocellulose with 0.15 M KC1. The polymerase was purified about 40-fold and was shown to be totally dependent on exogenous RNA for activity and relatively free of contaminating nuclease. The partially purified polymerase was able to use purified polio virion RNA as well as a template. Under the reaction conditions used, the polymerase required an oligouridylic acid primer and all four ribonucleside triphosphates for activity. The optimum ratio of oligouridylic acid molecules to poliovirus RNA molecules for priming activity was about 16:1. A nearest-neighbor analysis of the in vitro RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA product shows it to be heteropolymeric. Annealing the in vitro product with poliovirus RNA rendered it resistant to RNase digestion, thus suggesting that the product RNA was complementary to the virion RNA template.     

Novel properties of DNA polymerase beta with poly(rA).oligo(dT) template-primer.

Purified DNA polymerase beta of calf thymus can utilize poly(rA).oligo(dT) as efficiently as poly(dA).oligo(dT) or activated DNA as a template primer. The poly(rA).oligo(dT)-dependent activity of DNA polymerase beta was found to differ markedly from the DNA-dependent activity of the same enzyme (with either activated calf thymus DNA or poly(dA).(dT)10) in the following respects. 1) Poly(rA)-dependent activity was strongly inhibited by natural DNA from various sources or synthetic deoxypolymer duplexes at very low concentrations (less than 0.5 microgram/ml) at which the DNA-dependent activity was affected to a much smaller extent, if at all. 2) Poly(rA)-dependent activity was inhibited by N-ethylmaleimide more strongly than DNA-dependent activity measured at 37 degrees C, while it was resistant to this reagent at 26 degrees C. 3) The curves of the activity versus substrate concentration were sigmoidal in the poly(rA)-dependent reaction but hyperbolic in the activated DNA-dependent reaction. A kinetic study suggested that the association of beta-enzyme protomers may be required to copy the poly(rA) strand.     

Isolation and Properties of the Replicase of Encephalomyocarditis Virus

The RNA-dependent RNA polymerase (replicase) of encephalomyocarditis (EMC) virus was found to be closely associated with the smooth membranes of infected BHK-21 cells. An RNA-dependent EMC replicase was extracted from the membranes with 0.15% sodium dodecyl sulfate (SDS) and 1,1,2-trichlorotri-fluoroethane (Genetron 113) and further purified by high-salt dextran-polyethylene glycol phase separation, sievorptive chromatography, and glycerol gradient sedimentation. The enzyme does not manifest strict specificity toward EMC RNA template. It can use also Qbeta RNA, rRNA of BHK cells, or poly(C). SDS-polyacrylamide gel electrophoresis of purified EMC replicase labeled with radioactive methionine revealed that, of all the stable EMC proteins, the enzyme contains predominantly the 56,000-dalton (E) polypeptide.