Modifications of major aspects of myocardial ribonucleic acid metabolism as a response to noradrenaline. Action of the hormone on cytoplasmic processing of ribonucleic acid after reserpine treatment.

Treatment of perfused rabbit heart with reserpine causes a decrease of incorporation of labelled precursors into RNA species of subcellular fractions and polyamines. Ornithine decarboxylase, S-adenosylmethionine decarboxylase and cytoplasmic Mn2+-stimulated polyadenylate polymerase activities are not modified. Addition of noradrenaline to reserpine-treated perfused hearts enhances, compared with the control, the incorporation of precursor into RNA in all subcellular fractions other than the nuclear one, restores incorporation of labelled putrescine into polyamines, enhances ornithine decarboxylase and S-adenosylmethionine decarboxylase activities and causes a 12-fold increase in cytoplasmic Mn2+-dependent polyadenylate polymerase activity. After treatment with noradrenaline the increase in radioactivity was found solely in AMP after hydrolysis of microsomal RNA to nucleoside monophosphates.     

The role of subunits in yeast DNA-dependent ribonucleic acid polymerase A.

The properties of RNA polymerase A, which lacked the subunits of 48 000, 37 000 and 16 000 mol. wt., were compared with those of RNA polymerase A by using native calf thymus DNA as the template. The results showed that: (1) the specific activity of RNA polymerase A was about one-third that of RNA polymerase A; (2) more than 80% of RNA polymerase A, but only about 25% of RNA polymerase A, made RNA; (3) initiation by RNA polymerase A, but not by RNA polymerase A, began after a lag of 2 min; (4) the temperature-dependence for productive binding to DNA was greater for RNA polymerase A; (5) the apparent Km for UTP was greater for RNA polymerase A. These results support the supposition that the subunits missing from RNA polymerase A are involved in DNA binding [Huet, Dezélée, Iborra, Buhler, Sentenac & Fromageot (1976) Biochimie 58, 71-80] and show also that the loss of these subunits affects the elongation reaction.     

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

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.     

Convolution analysis of transcription by yeast DNA-dependent ribonucleic acid polymerase A. A mathematical method for studying ribonucleic acid chain elongation.

The rate of initiation of RNA synthesis catalysed by yeast RNA polymerase A on native calf thymus DNA decayed exponentially with a half-life of about 4.3 min. The rate constant for initiation was unaffected by preincubating the enzyme with DNA, or by decreasing the concentration of GTP 4-fold. The rate of RNA synthesis was constant for 15--20 min and then decreased. Each enzyme molecule made no more than one RNA molecule. In this situation, initiation, elongation and total RNA synthesis are related by a convolution integral. Solution of the convolution integral revealed that the rate of elongation was apparently biphasic. Analysis of the size of the RNA product showed that this biphasic profile arose because most but not all of the enzyme stopped RNA synthesis soon after initiation.     

The effect of feeding with a tryptophan-free amino acid mixture on rat liver magnesium ion-activated deoxyribonucleic acid-dependent ribonucleic acid polymerase

1. The Widnell & Tata (1966) assay method for Mg(2+)-activated DNA-dependent RNA polymerase was used for initial-velocity determinations of rat liver nuclear RNA polymerase. One unit (U) of RNA polymerase was defined as that amount of enzyme required for 1 mmol of [(3)H]GMP incorporation/min at 37 degrees C. 2. Colony fed rats were found to have a mean RNA polymerase activity of 65.9muU/mg of DNA and 18h-starved rats had a mean activity of 53.2muU/mg of DNA. Longer periods of starvation did not significantly decrease RNA polymerase activity further. 3. Rats that had been starved for 18h were used for all feeding experiments. Complete and tryptophan-deficient amino acid mixtures were given by stomach tube and the animals were killed 15-120min later. The response of RNA polymerase to the feeding with the complete amino acid mixture was rapid and almost linear over the first hour of feeding, resulting in a doubling of activity. The activity was still elevated above the starvation value at 120min after feeding. The tryptophan-deficient amino acid mixture produced a much less vigorous response about 45min after the feeding, and the activity had returned to the starvation value by 120min after the feeding. 4. The response of RNA polymerase to the feeding with the complete amino acid mixture was shown to occur within a period of less than 5min to about 10min after the feeding. 5. Pretreatment of the animals with puromycin or cycloheximide was found to abolish the 15min RNA polymerase response to the feeding with the complete amino acid mixture, but the activity of the controls was unaffected. 6. The characteristics of the RNA polymerase from 18h-starved animals and animals fed with the complete or incomplete amino acid mixtures for 1h were examined. The effects of Mg(2+) ions, pH, actinomycin D and nucleoside triphosphate omissions were determined. The [Mg(2+)]- and pH-activity profiles of the RNA polymerase from the animal fed with the complete mixture appeared to differ from those of the enzyme from the other groups, but this difference is probably not significant. 7. [5-(3)H]Orotic acid incorporation by rat liver nuclei in vivo was shown to be affected by the amino acid mixtures in a similar manner to the RNA polymerase. 8. The tryptophan concentrations of plasma and liver were determined up to 120 min after feeding with the amino acid mixtures. Feeding with the complete mixture produced a rapid increase in free tryptophan concentrations in both plasma and liver, but feeding with the incomplete mixture did not alter the plasma concentration. The liver tryptophan concentration increased at about 45min after feeding with the tryptophan-deficient diet. 9. There was a good correlation between the liver tryptophan concentration and RNA polymerase activity in all groups of animals. 10. It was concluded that the rat liver nucleus responded to an increase in amino acid supply by increased synthesis of RNA as a result of synthesis of RNA polymerase de novo. The correlation of tryptophan concentration and RNA polymerase activity appears to reflect the general amino acid concentration required to support hepatic protein synthesis and to produce new RNA polymerase. This new polymerase appears to differ from the basal RNA polymerase by its rapid synthesis and destruction, which may be a means of regulating RNA synthesis by the amino acid concentration in the liver.     

Nucleic acid synthesis and ribonucleic acid polymerase specificity in germinating and outgrowing spores of Bacillus subtilis.

Nucleic acid synthesis was studied during germination and outgrowth of normal spores of Bacillus subtilis, as well as of spores carrying the genome of phage phie. In a system in which development was restricted to the spore-darkening phase, synthesis of ribonucleic acid (RNA), but not deoxyribonucleic acid (DNA), was detected. The extent of RNA synthesis and turnover, during this phase was similar for the two types of spores. In a partially darkened population of spores of either type, there was little RNA degradation, whereas there was considerable turnover in a fully darkened population. The DNA-dependent RNA polymerase of dormant or dark spores was not active in vitro with phi DNA as template, although a sigma-like factor could be separated from the polymerizing activity by zone centrifugation. Within 40 min after resuspension of dark spores in a medium that allows outgrowth, the enzyme acquired the ability to transcribe the phage DNA efficiently. During outgrowth, both normal and carrier spores synthesized DNA, but in carrier spores this DNA was almost entirely phage specific. The pattern of RNA accumulation in normal spores was in two distinct phase (0 to 60 min and 90 to 180 min). The second phase was absent in outgrowing carrier spores. The burst of phage in carrier spores occurred at 160 to 180 min.     

Further characterization of the effects of 3-methylcholanthrene administration upon hepatic ribonucleic acid polymerase activities

The administration of 3-methylcholanthrene (MC) to rats results in a marked increase in the specific activities of hepatic RNA polymerases I and II. In the present study, we were able to show that this increase was not caused by a shift in the ratio of ‘free’ to ‘template-engaged’ RNA polymerase. By means of binding studies with [³H]amatoxin, we were unable to demonstrate any increase in the number of RNA polymerase II molecules in liver after MC administration to the rats. RNA polymerase I was purified in excess of 3000-fold from hepatic nuclei isolated both from control and MC-treated rats. The stimulation in activity was demonstrated at each step in the purification scheme until glycerol sedimentation analysis. Results from cation-exchange chromatography on phosphocellulose indicated that the polycyclic hydrocarbon increased the enzyme activity of RNA polymerase I_b somewhat specifically. Subsequent to glycerol gradient centrifugation, this stimulatory advantage was no longer evident. Reconstitution experiments revealed the presence of a stimulatory component, which was demonstrated in low molecular weight fractions from both control and experimental preparations.     

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

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].     

Evidence for stable messenger ribonucleic acid during sporulation and enterotoxin synthesis by Clostridium perfringens type A.

Stable messenger ribonucleic acid (mRNA) was shown to be involved in both enterotoxin synthesis and synthesis of other spore coat proteins in Clostridium perfringens. When used at a concentration that inhibited [14C]uracil incorporation, rifampin, a specific inhibitor of deoxyribonucleic acid-dependent RNA polymerase, prevented incorporation of a mixture of labeled amnoo acids by 3-h sporulating cells. At that time, enterotoxin protein was first detectable and cells were primarily at stage II or III of sporulation. When rifampin or streptolydigin was added to 5-h sporulating cells, which were primarily at stage IV or V and had significant toxin levels, incorporation of labeled amino acids continued through 30 min despite its presence. Rifampin also failed to prevent the specific synthesis of enterotoxin, a structural protein of the spore coat. The half-life of enterotoxin RNA was estimated to be at least 58 min. When cell extracts from 5-h sporulating cells that had been exposed to 3H-labeled amino acids for 10 min were subjected to electrophoresis on polyacrylamide gels and the gels were subsequently analyzed for radioactivity, two major peaks of radioactivity were obtained. The two peaks corresponded to enterotoxin and another spore coat protein(s). Similar results were obtained when the cells had been preincubated for 60 min with rifampin before label addition, indicating the functioning of stable mRNA.     

Effect of actinomycin D and guanidine on the formation of a ribonucleic acid polymerase induced by foot-and-mouth-disease virus and on the replication of virus and viral ribonucleic acid

The RNA-dependent RNA polymerase induced in baby-hamster kidney cells by infection with foot-and-mouth-disease virus can be detected as early as 60min. after infection, which is 60min. before viral RNA synthesis commences. The time at which the polymerase can first be detected coincides with the latest time at which actinomycin D (50mug./10(7) cells) or guanidine (1mg./10(7) cells) inhibits virus replication. However, by increasing the concentration of guanidine, viral replication can be inhibited later in the growth cycle, casting doubt on the validity of the hypothesis that guanidine acts specifically on the formation of the viral RNA polymerase.     

The inhibition of ribonucleic acid polymerase by acridines

1. The aminoacridines, proflavine (3,6-diaminoacridine) and 9-aminoacridine, and a hydrogenated derivative, 9-amino-1,2,3,4-tetrahydroacridine, were shown to inhibit in vitro the DNA-primed RNA polymerase of Escherichia coli. The inhibition is strong with both proflavine and 9-aminoacridine, but weak with 9-amino-1,2,3,4-tetrahydroacridine. 2. The extent to which the three acridines bind to calf-thymus DNA in the enzyme medium was studied spectrophotometrically. The extent of binding decreases in the order: proflavine, 9-aminoacridine, 9-amino-1,2,3,4-tetrahydroacridine. Some evidence was also obtained for interaction between the nucleoside triphosphate substrates and proflavine or 9-aminoacridine; no such interaction was detectable with 9-amino-1,2,3,4-tetrahydroacridine. 3. Although the amount of acridine bound to DNA increases with increasing inhibition, a stage is reached where an increase in acridine concentration still causes an increase in inhibition, with practically no increase in the amount bound to DNA. 4. Plots of reciprocal rates against the reciprocal of DNA concentration were linear and had a common intercept when proflavine or 9-aminoacridine was present. Similar relations were obtained when the reciprocal concentration of nucleoside triphosphates was plotted. The observations are interpreted kinetically in terms of a competitive inhibition of the enzyme by proflavine or 9-aminoacridine and of a kinetic role for the DNA analogous to ;activation'. 5. This suggests that inhibitory acridine molecules can occupy the sites on the RNA polymerase that are specific for binding the nucleoside triphosphate substrate or the bases of the DNA, when these become accessible during the copying process.     

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.     

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.     

Disappearance of radioactivity from the various ribonucleic acid pools and acid-soluble fractions of mouse liver and kidney after a single injection of labelled orotic acid. The effect of castration

1. An attempt was made to study the rate of synthesis as well as the distribution of RNA in the various cellular fractions in the livers and kidneys of normal and castrated mice. 2. The tissue was fractionated by the procedure of Blobel & Potter (1967). By using this method it was not possible to find any pronounced difference in the relative proportions of RNA in isolated subcellular fractions when kidneys from normal and castrated mice were compared. On the other hand there was an indication of a shift toward the bound ribosomes in livers from normal mice in comparison with livers from castrated mice. 3. Disappearance of the radioactivity followed the pattern of the first-order reaction. Comparing the half-lives of RNA in liver and kidneys it was found that in the latter in both groups of animals half-lives were shorter no matter which cellular fraction was studied. 4. The half-lives for total homogenate RNA, total ribosomal RNA and low-molecular-weight RNA from kidneys of castrated mice were approximately 20-25% longer than the half-lives for the corresponding fractions from normal mouse kidneys. 5. An explanation is put forward for the anomalous finding that RNA from the castrated-mouse kidneys has a higher specific radioactivity than that isolated from normal mice.     

Rapid micromethod for the purification of Escherichia coli ribonucleic acid polymerase and the preparation of bacterial extracts active in ribonucleic acid synthesis.

A rapid micromethod is described for the preparation of nucleic acid-free extracts from Escherichia coli that involves precipitation with polyethylene glycol. Extracts can be prepared from growing cells in 75 min by three short, low-speed centrifugations. The extract did not inhibit added purified ribonucleic acid (RNA) polymerase, suggesting that major inhibitors of RNA synthesis had been removed. This extract should be ideal for assessing the properties of mutant RNA polymerases. The rapid chromatography of the extracts with step elution from deoxyribonucleic acid- and diethylaminoethyl-cellulose columns resulted in high yields of substantially pure RNA polymerase. We used this technique to purify 35S-labeled RNA polymerase. This system should find application for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase.