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

ABSTRACT: 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.

SUBMITTER: Louis BG

PROVIDER: S-EPMC1173895 | biostudies-other | 1972 Jul

REPOSITORIES: biostudies-other

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

Project description:1. The subunits alpha and beta 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, alphabeta, in the absence of salt is due to the rapid inactivation of the beta 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. T(7) DNA is transcribed asymmetrically and the enzyme selectively copies the T(7) ;early' genes. 8. Preliminary amino acid analyses of alpha and beta subunits show that their overall content of acidic, basic and neutral amino acids does not differ appreciably from that of Escherichia coli RNA polymerase.

| S-EPMC1178560 | biostudies-other

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

Project description: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.

| S-EPMC1176639 | biostudies-other

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

Project description: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.

| S-EPMC1176638 | biostudies-other

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

Project description: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.

| S-EPMC1178561 | biostudies-other

Halobacterium cutirubrum ribosomes. Properties of the ribosomal proteins and ribonucleic acid.

Project description:1. The 30S ribosomal subunit of the extreme halophile Halobacterium cutirubrum is unstable and loses 75% of its ribosomal protein when the 70S ribosome is dissociated into the two subunits. A stable 30S subunit is obtained if the dissociation of the 70S particle is carried out in the presence of the soluble fraction. 2. A fractionation procedure was developed for the selective removal of groups of proteins from the 30S and 50S subunits. When the ribosomes, which are stable in 4m-K(+) and 0.1m-Mg(2+), were extracted with low-ionic-strength buffer 75-80% of the 30S proteins and 60-65% of the 50S proteins as well as the 5S rRNA were released. The proteins in this fraction are the most acidic of the H. cutirubrum ribosomal proteins. Further extraction with Li(+)-EDTA releases additional protein, leaving a core particle containing either 16S rRNA or 23S rRNA and about 5% of the total ribosomal protein. The amino acid composition, mobility on polyacrylamide gels at pH4.5 and 8.7, and the molecular-weight distribution of the various protein fractions were determined. 3. The s values of the rRNA are 5S, 16S and 23S. The C+G contents of the 16S and 23S rRNA were 56.1 and 58.8% respectively and these are higher than C+G contents of the corresponding Escherichia coli rRNA (53.8 and 54.1%).

| S-EPMC1174305 | biostudies-other

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

Project description: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.

| S-EPMC1165679 | biostudies-other

Partial purification and properties of Halobacterium cutirubrum L-alanine dehydrogenase.

Project description:1. Halobacterium cutirubrum L-alanine dehydrogenase was purified approx. 100-fold. 2. It has a mol. wt. of 72 500, about one-third that of two well-studied alanine dehydrogenases from non-halophiles. 3. The activity of the enzyme increases with temperature up to 70 degrees C, but the protein itself is not thermostable. 4. In the reductive amination reaction, the enzyme is fully active in the presence of high concentrations of K+, Na+ or NH4+ and partially active with Cs+ or Li+, but for oxidative deamination it has an absolute requirement for K+.

| S-EPMC1164509 | biostudies-other

Nucleic acid enzymology of extremely halophilic bacteria. Gel-filtration and density-gradient-centrifugation studies of the molecular weights of Halobacterium cutirubrum polynucleotide phosphorylase and deoxyribonucleic acid- and ribonucleic acid-dependent ribonucleic acid polymerases.

Project description: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.

| S-EPMC1176640 | biostudies-other

A procedure for the rapid purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase.

Project description:A rapid procedure involving DNA-cellulose chromatography followed either by sedimentation in a high-salt glycerol gradient or by gel filtration is described for the complete purification of Escherichia coli DNA-dependent RNA polymerase.

| S-EPMC1177684 | biostudies-other

Purification and properties of a nuclear protein kinase associated with ribonucleic acid polymerase I.

Project description:A cyclic AMP-dependent nuclear protein kinase was found to be closely associated with rat liver nucleolar RNA polymerase I throughout most of its purification. This protein kinase was purified to near homogeneity. It exhibits a number of unusual catalytic properties, including the inability to utilize Mn2+ when RNA polymerase is the substrate and the ability to phosphorylate both acidic and basic substrates. Phosphorylation of RNA polymerase I by this protein kinase results in the formation of phosphoester bonds characteristic of phosphoserine and phosphothreonine. Radioautography of polyacrylamide-gel electrophoretograms of the phosphorylated RNA polymerase I revealed that the 32P was located primarily on enzyme subunits SA1, SA3, SA5, and SA6 [nomenclature of Kedinger, Gissinger & Chambon (1974) Eur. J. Biochem, 44, 421-436].

| S-EPMC1184174 | biostudies-other

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