Methylation of newly synthesized ribonucleic acid by isolated rat liver nuclei. Characterization of the ribonucleic acid synthesized by nuclei from starved animals

1. Nuclei from rat liver incubated with S-adenosyl[methyl-(14)C]methionine incorporated radioactivity into RNA and into lipid and protein. 2. All of the labelled RNA was extracted from the nuclei with trichloroacetic acid at 90 degrees C. 3. The [(14)C]methyl-group incorporation into the hot-trichloroacetic acid extract was 30% inhibited by the addition of actinomycin D (100mug/mg of DNA) or by the omission of CTP, GTP and UTP. 4. Assuming that the main substrate for this triphosphate-dependent methylation was newly synthesized precursor rRNA containing one methyl group/30 uridylate residues, it was calculated that approx. 60% of the [(14)C]UMP incorporated under similar conditions represented precursor rRNA synthesis. 5. In agreement with this, low concentrations of actinomycin D (approx. 1mug/mg of DNA) sufficient to abolish the triphosphate-dependent incorporation of [(14)C]methyl group inhibited 68% of the [(14)C]UMP incorporation. 6. The incorporation of [(14)C]UMP by nuclei from starved animals decreased progressively with increasing periods of starvation, whereas the triphosphate-dependent [(14)C]methyl-group incorporation was not further decreased after 1 day of starvation. 7. This suggests that precursor rRNA synthesis decreased within 1 day whereas other species of RNA were affected only after longer periods of starvation.     

Effect of Tobacco Mosaic Virus Infection on Amino Acid Incorporation into Isolated Mitochondria from Tobacco Leaves

Mitochondria isolated from tobacco leaves incorporated ¹⁴C-leucine into the protein and the rate was enhanced by tobacco mosaic virus (TMV) infection as compared with noninfected level. In vitro amino acid incorporation by mitochondria required adenosine triphosphate (ATP), Mg²⁺, and KC1 and the energy sources from oxidative phosphorylation as well as from ATP-generating system. This incorporation was inhibited by ribonuclease (RNase), deoxyribonuclease (DNase), actinomycin D, mitomycin C, puromycin, and chloramphenicol added in the reaction medium. The pretreatment of the mitochondria with DNase and actinomycin D reduced the rate of incorporation. The mitochondria incorporated ³H-guanosine triphosphate (GTP) and this activity was blocked by actinomycin D. The presence in this system of 15,000 g supernatant cell sap fraction or bacterial contamination was carefully checked obtaining a negative result. The reaction product into which ^l4C-amino acids incorporated was solubilized by trypsin. The nature of the amino acid incorporating activity of isolated mitochondria obtained from TMV-infected tobacco leaves is discussed.     

Chain extension of ribonucleic acid by enzymes from rat liver cytoplasm

1. The 105000g supernatant fraction of rat liver catalyses the incorporation of ribonucleotides from ribonucleoside triphosphates into polyribonucleotide material. The reaction requires Mg²⁺ ions and is enhanced by the addition of an ATP-generating system and RNA, ATP, UTP and CTP but not GTP are utilized in this reaction. In the case of UTP, the product is predominantly a homopolymer containing 2–3 uridine residues, and there is evidence that these may be added to the 3′-hydroxyl ends of RNA or oligoribonucleotide primers. 2. The microsome fraction of rat liver incorporates ribonucleotides from ATP, GTP, CTP and UTP into polyribonucleotide material. This reaction requires Mg²⁺ ions and is enhanced slightly by the addition of an ATP-generating system, and by RNA but not DNA. Supplementation of the reaction mixture with the three complementary ribonucleoside 5′-triphosphates greatly increases the utilization of a single labelled ribonucleoside 5′-triphosphate. The optimum pH is in the range 7·0–8·5, and the reaction is strongly inhibited by inorganic pyrophosphate and to a much smaller degree by inorganic orthophosphate. It is not inhibited by actinomycin D or by deoxyribonuclease. In experiments with [³²P]UTP in the absence of ATP, GTP and CTP, 80–90% of ³²P was recovered in UMP-2′ or -3′ after alkaline hydrolysis of the reaction product. When the reaction mixture was supplemented with ATP, GTP and CTP, however, about 40% of the ³²P was recovered in nucleotides other than UMP-2′ or -3′. Although the reactions seem to lead predominantly to the synthesis of homopolymers, the possibility of some formation of some heteropolymer is not completely excluded.     

In situ regulation of mammalian CTP synthetase by allosteric inhibition

The regulatory role of the allosteric site of CTP synthetase on flux through the enzyme in situ and on pyrimidine nucleotide triphosphate (NTP) pool balance was investigated using a mutant mouse T lymphoblast (S49) cell line which contains a CTP synthetase refractory to complete inhibition by CTP. Measurements of [3H]uridine incorporation into cellular pyrimidine NTP pools as a function of time indicated that CTP synthesis in intact wild type cells was markedly inhibited in a cooperative fashion by small increases in CTP pools, whereas flux across the enzyme in mutant cells was much less affected by changes in CTP levels. The cooperativity of the allosteric inhibition of the enzyme was greater in situ than in vitro. Exogenous manipulation of levels of GTP, an activator of the enzyme, indicated that GTP had a moderate effect on enzyme activity in situ, and changes in pools of ATP, a substrate of the enzyme, had small effects on CTP synthetase activity. The consequences of incubation with actinomycin D, cycloheximide, dibutyryl cyclic AMP, and 6-azauridine on the flux across CTP synthetase and on NTP pools differed considerably between wild type and mutant cells. Under conditions of growth arrest, an intact binding site for CTP on CTP synthetase was required to maintain a balance between the CTP and UTP pools in wild type cells. Moreover, wild type cells failed to incorporate H14CO3- into pyrimidine pools following growth arrest. In contrast, mutant cells incorporated the radiolabel at a high rate indicating loss of a regulatory function. These results indicated that uridine nucleotides are important regulators of pyrimidine nucleotide synthesis in mouse S49 cells, and CTP regulates the balance between UTP and CTP pools.     

Initiation of RNA synthesis in isolated rat liver nuclei

Isolated rat liver nuclei were incubated under conditions when RNA polymerase I or RNA polymerase II was preferentially active. It was shown that [gamma-32P] ATP and [gamma-32P] GTP were incorporated into phenol extractable, TCA-precipitable material. RNase, actinomycin D, heparin and, in the case of RNA-polymerase II, alpha-amanitine inhibited precursor incorporation. These data are interpreted as evidence in favour of the initiation of RNA synthesis in isolated rat liver nuclei.     

Polyriboadenylate synthesizing activity in chromatin of wheat seedlings

The occurrence of poly(A) polymerase was demonstrated in chromatin of wheat seedlings. The optimum pH was about 8.0. The activity was depressed by the treatment of chromatin with pronase or deoxyribonuclease, and it was sensitive to ribonuclease. AMP, ADF, inorganic pyrophosphate, CTP, GTP, UTP, dATP, dCTP, dGTP, dTTP, and lysine inhibited the activity, while aspartate stimulated it. Rifampicin significantly, and actinomycin D insignificantly inhibited the activity. Difference of the susceptibilities to dTTP and to tryptophane indicates that poly(A) polymerase is unlike RNA polymerase.     

The synthesis of polyribonucleotides by cytoplasmic enzymes

1. The possibility that the cell cytoplasm contains enzymes catalysing the biosynthesis of RNA was investigated in fractions obtained by differential centrifugation of homogenates of Landschutz ascites-tumour cells. 2. The microsomal fraction was shown to be most active in incorporating UMP residues from [alpha-(32)P]UTP into polyribonucleotide material. 3. The same fraction also incorporated [(3)H]CTP, [(3)H]ATP and [(3)H]GTP separately and independently of the presence of complementary ribonucleoside 5'-triphosphates. 4. The reaction was promoted by the addition of RNA and showed an absolute requirement for Mg(2+) ions. 5. Analysis of alkaline hydrolysates of the reaction products after the incorporation of [alpha-(32)P]UTP showed that most of the radioactivity was recovered in (2',3')-UMP residues irrespective of whether CTP, ATP and GTP were present in the reaction mixture. 6. Extraction of RNA from the reaction mixtures after the incorporation of [(3)H]ATP, [(3)H]GTP or [(3)H]CTP and analysis by sucrosedensity-gradient centrifugation showed no labelling of the ribosomal RNA. Radioactive material appeared between the 4s region and the meniscus of the sucrose gradient. In agreement with this observation, determinations of the chain length of the product showed that only short sequences of polynucleotides were synthesized. It is concluded that only homopolyribonucleotide synthesis is catalysed by the microsomal fractions and that there is little or no synthesis of RNA-like heteropolymers.     

New ATP incorporating activities from rat liver mitochondria: the attachment of a portion of ATP to a pronase-sensitive receptor

Studies using a Brij 58 detergent extract of rat liver mitochondria reveal that these organelles can catalyze the time-dependent incorporation of a portion of [³H]ATP into an acid-insoluble product. The activities studied using 8 mM Mn⁺⁺ or 15 mM Mg⁺⁺ are stimulated by dithiothreitol and by CTP, GTP or UTP, while that studied using 2 mM Mg⁺⁺ is not. The incorporated tritium remains bound after incubation in the presence of excess unlabeled ATP and chromatography on Sephadex G-25. The labeled product is insensitive to ribonuclease A and snake venom phosphodiesterase, but is sensitive to pronase. The attached portion of the ATP molecule released upon treatment of the product has been tentatively identified as adenosine for the activities studied using 2 mM Mg⁺⁺ or 8 mM Mn⁺⁺ and as AMP (80%) and adenosine (20%) for the reaction studied using 15 mM Mg⁺⁺.     

Properties of unprimed poly(A)-poly(U) synthesis by Caulobacter crescentus RNA polymerase.

Some properties of unprimed poly(A)-poly(U) synthesis by DNA-dependent RNA polymerase from Caulobacter crescentus were examined. The reaction required ATP and UTP as substrates and manganese as a divalent cation. Rifampicin completely inhibited the reaction at a concentration of 1 micron/ml, and the enzyme catalyzed the polymer synthesis well regardless of the presence of GTP, CTP or both. The chain length of the poly(A)-poly(U) synthesized was about one hundred base pairs, as estimated from a sedimentation velocity and the molar ratio of [3H]AMP to [gamma-32P]ATP incorporated into the poly(A)-poly(U). The reaction was dependent on the square of the enzyme concentration and the enzyme dimers formed complexes with poly(A)-poly(U) during the reaction.     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

Inhibitory effects of nucleoside triphosphates on nucleolar RNA synthesis.

Studies on the effects of substrates on RNA polymerase I [EC 2.7.7.6] in vitro showed that nucleolar RNA synthesis was inhibited by an excess of substrate nucleoside triphosphates in the presence of Mg2+. GTP and UTP were more inhibitory than CTP and ATP. These compounds specfically inhibited nucleolar RNA synthesis and a concentration of GTP that strongly inhibited nucleolar RNA synthesis did not inhibit RNA synthesis by partially purified RNA polymerase I. The inhibition of nucleolar RNA synthesis disappeared at pH 9.0 without any change in the apparent Km for GTP or the Vmax of RNA synthesis.     

RNA polymerase activity in double-stranded ribonucleic acid virus particles from Aspergillus foetidus.

RNA polymerase activities have been detected in purified particles of $\text{Aspergillus foetidus}$ viruses S and F. Incorporation of [³H]-UTP into acid insoluble RNA was dependent on ATP, GTP, CTP and magnesium ions. No pretreatment of the particles was required and the rate of reaction was proportional to the amount of virus added. In the conditions used RNA synthesis by $\text{A. foetidus}$ virus S was complete in 4 h. The reaction could be stimulated by Triton X-100, but was unaffected by heat shock, dithiothreitol, potassium chloride or ammonium chloride; it was inhibited by ethidium bromide but not by actinomycin D. The major reaction product was single-stranded RNA, as indicated by its sensitivity to degradation by ribonuclease A. This is the first report of synthesis of single-stranded RNA by a double-stranded RNA mycovirus.     

In organello transcription in maize mitochondria and its sensitivity to inhibitors of RNA synthesis

Purified mitochondrial preparations from etiolated maize shoots support the incorporation of radioactivity from labeled UTP into RNA. The incorporation is linear with time for up to 2 hours, shows Michaelis-Menton kinetics with respect to the concentration of the labeled substrate, UTP, and has salt and pH optima which are different than those previously reported for RNA synthesis by isolated chloroplasts. When a crude mitochondrial preparation is subjected to isopycnic sucrose gradient centrifugation, the bulk of the RNA synthetic activity co-sediments with mitochondrial marker enzymes and with the mitochondrial 26S and 18S rRNAs. Maize mitochondrial RNA synthesis is prevented by actinomycin D and ethidium bromide but unaffected by α-amanitin. It is strongly inhibited by rifampicin at concentrations which have no effect on nuclear and chloroplast RNA synthesis, but only moderately inhibited by rifampicin at concentrations which completely inhibit bacterial RNA synthesis. The optimization, cell fractionation, and inhibitor data all suggest that contaminating organelles and bacteria do not contribute appreciably to the RNA synthesis in purified mitochondrial preparations.     

Biochemistry of terminal deoxynucleotidyltransferase: mechanism of inhibition by adenosine 5'-triphosphate

The polymerization of deoxyribunucleoside triphosphate catalyzed by terminal deoxyribonucleotidyltransferase (TdT, EC 2.7.7.31) is severely inhibited by the addition of ribonucleoside triphosphates, ATP being the most potent inhibitor. Examination of the inhibitory effect of ATP using oligo(dA)12-18 as well as activated DNA as primers revealed that (a) ATP inhibition is not due to its addition onto a 3'-OH primer terminus ad judged by the lack of incorporation of labeled ATP, although under similar conditions incorporation of GTP can be demonstrated, (b) a consistent degree of inhibition was noted independent of primer or enzyme concentration; (c) addition of ATP to an ongoing reaction promptly reduces the rate of polymerization; (d) kinetic studies indicate a competitive (with respect to substrate deoxy triphosphate) pattern of inhibition; (e) addition of excess deoxyribotriphosphate promptly relieves the inhibition. Unlike ATP, other ribotriphosphates yield a mixed pattern of inhibition partly mediated by competitive mechanisms. GTP and CTP and to a minor extent UTP are incorporated into DNA in the presence or absence of deoxy triphosphate. Furthermore, addition of ATP also inhibits incorporation of GTP and CTP.     

Incorporation of amino acids into protein by cell-free extracts of penicillium chrysogenum

Summary The preparation of ribosomal particles from P. chrysogenum has been described. After correction to 20°C and extrapolation to infinite dilution, they had sedimentation coefficients of approximately 80S, 60 S, and 40 S. The washed ribosomes plus purified supernatant fraction, KCl and sucrose incorporated C¹⁴-l-amino acids into protein. There was no stimulation of incorporation by ATP, GTP, CTP, UTP, or Mg ions. Incorporation was inhibited by streptomycin and chloramphenicol but not by ribonuclease.The amino acid incorporating system from P. chrysogenum did not resemble bacterial and yeast systems in many respects and reasons for this are discussed.     

Effect of nucleotide structure on cardiac myosin subfragment 1 transient kinetics

Transient kinetic data of the hydrolysis of several nucleotides (TTP, CTP, UTP, GTP) by cardiac myosin subfragment 1 (S1) were analyzed to obtain values for the equilibrium constant for nucleotide binding and rate constants for the S1-nucleotide isomerization and the subsequent nucleotide hydrolysis as well as the magnitudes of the relative fluorescence enhancements of the myosin that occur upon isomerization and hydrolysis. These data are compared with data from a previous study with ATP. Nucleotide binding is found to be relatively insensitive to nucleotide ring structure, being affected most by the group at position C6. Isomerization and hydrolysis are more sensitive to nucleotide structure, being inhibited by the presence of a bulky group at position C2. Kinetic parameters decrease as follows: for binding, GTP greater than UTP approximately TTP greater than ATP greater than CTP; for isomerization, ATP greater than UTP approximately TTP approximately CTP greater than GTP; for hydrolysis, ATP greater than TTP greater than CTP approximately UTP greater than GTP. Fluorescence enhancements appear to be most dependent upon the relative values of the individual rate constants.