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

ABSTRACT: 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.

SUBMITTER: Louis BG

PROVIDER: S-EPMC1178561 | biostudies-other | 1972 Mar

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 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

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

Project description: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.

| S-EPMC1173895 | 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

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

Evidence for the lack of deoxyribonucleic acid dark-repair in Halobacterium cutirubrum.

Project description:1. Halobacterium cutirubrum does not perform dark-repair of DNA either after u.v. irradiation or during normal growth. 2. Cultures irradiated with u.v. are readily photoreactivated, but do not recover viability in the dark. 3. No increase in the rate of DNA synthesis is observed in the surviving cells after u.v. irradiation. 4. At early times during normal semiconservative replication, newly incorporated thymidine is found only in the hybrid DNA. 5. It is suggested that these bacteria may be useful in the study of DNA replication and photoreactivation.

| S-EPMC1163790 | 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

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

Deoxyribonucleic acid-dependent ribonucleic acid polymerase activity in rat liver after protein restriction.

Project description:Rats were fed for 6 days on a diet containing either 3 or 20% high-quality protein. Nuclei were isolated from liver and DNA-dependent RNA polymerases (EC 2.7.7.6) extracted with 1 M-(NH4)2SO4. The proteins were then precipitated with 3.5 M-(NH4)2SO4 and after dialysis applied to a DEAE-Sephadex column. The column was developed with a gradient of (NH4)2SO4. Polymerase I separated well from alpha-amanitin-sensitive polymerase II. The enzyme activities were compared between the two dietary groups. Rats that had received 3% protein showed a lower polymerase I activity per g wet wt. of liver, per mg of DNA and per mg of protein. Polymerase II was lower in activity per g wet wt. of liver and per mg of DNA, but was higher per mg of protein. Polyacrylamide-gel electrophoretograms showed a higher proportion of contaminating proteins in polymerase II fractions isolated from 20%-protein-fed rats. The data explain the lower activity obtained per mg of protein in these rats. It is concluded that a decrease in dietary protein content from 20 to 3% induces a fall in content and specific activity of RNA polymerase I and II in liver.

| S-EPMC1165505 | biostudies-other

Contemporaneous isolation of deoxyribonucleic acid-dependent ribonucleic acid polymerase and poly(A) polymerase from rat liver mitochondria.

Project description:1. Poly(A) polymerase and DNA-dependent RNA polymerase from rat liver mitochondria can be completely separated by using two different chromatographic procedures. 2. Poly(A) polymerase can only incorporate ATP into acid-insoluble material and strongly depends on the addition of an endogenous factor (probably containing a mixture of oligoribonucleotides), but it is not stimulated by DNA. 3. RNA polymerase is fully DNA-dependent and rifampicin-sensitive, but was not stimulated by the endogenous factor mentioned above. 4. The chromatographic behaviour of the two enzymes, together with the properties described, suggest that they represent two different protein molecules.

| S-EPMC1163853 | biostudies-other

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