Mechanism of inhibition of Escherichia coli RNA polymerase by captan.

Captan (N-trichloromethylthiocyclohex-4-ene-1,2-dicarboximide) was shown to inhibit RNA synthesis in vitro catalysed by Escherichia coli RNA polymerase. Incorporation of [gamma-32P]ATP and [gamma-32P]GTP was inhibited by captan to the same extent as overall RNA synthesis. The ratio of [3H]UTP incorporation to that of [gamma-32P]ATP or of [gamma-32P]GTP in control and captan-treated samples indicated that initiation was inhibited, but the length of RNA chains being synthesized was not altered by captan treatment. Limited-substrate assays in which re-initiation of RNA chains did not occur also showed that captan had no effect on the elongation reaction. Studies which measured the interaction of RNA polymerase with template DNA revealed that the binding of enzyme to DNA was inhibited by captan. Glycerol-gradient sedimentation of the captan-treated RNA polymerase indicated that the inhibition of the enzyme was irreversible and did not result in dissociation of its subunits. These data are consistent with a mechanism in which RNA polymerase activity was irreversibly altered by captan, resulting in an inability of the enzyme to bind to the template. This interaction was probably at the DNA-binding site on the polymerase and did not involve reaction of captan with the DNA template.     

Enhancement of in vitro ribonucleic acid synthesis on chromium(III)-bound chromatin

Z chromatin-chromium (Cr) complex, prepared from mouse liver chromatin and CrCl3, showed a significantly enhanced template activity for in vitro RNA synthesis. Digestion experiments with this complex using micrococcal nuclease and DNase I suggested that Cr(III) preferentially binds to linker regions rather than core regions of chromatin. Further, it was found that Cr(III) binds to DNA and nonhistone proteins (NHP), but hardly to histones. Moreover, the template activity of an NHP-Cr complex, when added to a DNA-histones complex, was inhibited remarkably. The template activity of the chromatin-Cr complex was not significantly altered by proteinase K digestion. Furthermore, experiments using rifampicin and [gamma-32P]guanosine 5'-triphosphate (GTP) demonstrated an increase in the number of initiation sites in the chromatin-Cr complex. These results suggest that, in this in vitro system, Cr(III) preferentially binds to DNA in chromatin and causes an increase in the number of initiation sites, thus enhancing RNA synthesis.     

Studies on DNA content, RNA synthesis, and DNA template activity in aging cells of Paramecium aurelia.

Investigations by Feulgen microspectrophotometry in Paramecium aurelia indicated that as fission age increased the amount of macronuclear DNA decreased. It was also found that the amount of RNA synthesis as determined by the in vivo incorporation of [³H]uridine decreased as the fission age increased. An alternative in situ assay of the DNA template activity determined by the RNA polymerase-catalyzed incorporation of [³H]UTP is described. The DNA template activity of older cells was shown to be significantly lower on a per cell basis than that of younger cells. The majority of this reduction was shown to be due to the gradual loss of DNA template with an increase in fission age. The specific activity of the DNA template, however, does show a small but significant decrease as the fission age of the cell increases.     

Amino acid dependent and independent insulin stimulation of cartilage metabolism

The effects of insulin on embryonic chicken cartilage in organ culture and the dependence of these effects on essential amino acids have been studied. In the presence of all essential amino acids, insulin: (1) increases 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake; (2) increases [5(-3H] uridine flux into uridine metabolites and the intracellular UTP pool; (3) expands the size of the intracellular UTP pool; (4) does not change the specific activity of the UTP pool; and (5) stimulates RNA, proteoglycan, and total protein synthesis. In lysine (or other essential amino acid)-deficient medium, the effects of insulin are different. While insulin stimulates incorporation of [5(-3)H] uridine into RNA, it does so by increasing the specific activity of the UTP pool without increasing RNA synthesis. Insulin stimulates 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake but no longer stimulates proteoglycan, total protein, or RNA synthesis or expands the size of the UTP pool. These data indicate that there are amino acid dependent and independent effects of insulin on cartilage. Transport processes are amino acid independent, while synthetic processes are amino acid dependent.     

Display of 8-hydroxy-GTP substrate properties of UTP in the reaction of polynucleotide synthesis catalyzed by RNA-polymerase from Escherichia coli in the presence of poly[d(AT).d(AT)] template

8-oxy-GTP was obtained via reaction of GTP with ascorbic acid and addition of hydrogen peroxide. 8-oxy-GTP is recognized and displays substrate properties of UTP on substitution of 8-oxy-GTP for UTP in polynucleotide synthesis catalyzed by E. coli RNA polymerase on a poly[d(A-T)].poly[d(A-T)] template. Such incorporation does not take place at equimolar quantities of GTP and 8-Br-GTP. The incorporation of 8-oxy-GTP instead of UTP, is 2.5-3 times higher upon replacement of Mg2+ by Mn2+ ions. The dinucleotide ApU serving as an initiator rises the incorporation level of 8-oxy-GTP both for Mg2+ and Mn2+ ions. 8-oxy-GTP slightly inhibits poly[r(A-U)] synthesis, but UTP strongly inhibits the incorporation of 8-oxy-GTP. [alpha-32P] 8-oxy-GTP is incorporated mainly instead of UTP, but it can be incorporated also during the substitution of 8-oxy-GTP for ATP.     

Utilization of L-cell nucleoside triphosphates by Chlamydia psittaci for ribonucleic acid synthesis.

Long-term, 32-P-labeled L cells were infected with the obligately intracellular parasite Chlamydia psittaci (strain 6 BC). At 20 h postinfection, [3-H]uridine was added, and the infected cells were sampled at intervals for incorporation of the labels into the uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools of the host L cell and the uridine monophosphate (UMP) and cytidine monophosphate (CMP) in 16S ribosomal ribonucleic acid (RNA) of the parasite. The specific activity of the nucleotides was calculated from the ratio of 3-H to 32-P counts in the nucleotides. The rate of approach to equilibrium labeling of UTP and CTP in L-cell pools and UMP and CMP in 16S RNA from the exogenous uridine label was determined from the increase in the ratios of the specific activities of CTP to UTP and CMP to UMP with time. The rate of approach to equilibrium CMP:UMP labeling of the 16S RNA of C. psittaci was consistent with the rate predicted from the kinetics of labeling of the CTP and UTP pools of the host L cell. In analogous experiments, the rate of approach to equilibrium guanosine monophosphate:adenosine monophosphate labeling of 16S RNA from an exogenous [14-C]adenine label was consistent with the rate predicted from the kinetics of labeling of the purine nucleoside triphosphate pool of the host cell. These results support the concept that members of the genus Chlamydia owe their obligate intracellular mode of reproduction to a requirement for energy intermediates which is fulfilled by the host cell. In addition, evidence was obtained that the total acid-soluble purine nucleoside triphosphate pool of L cells accurately represents the precursors of L-cell 18S ribosomal RNA.     

Effects of 3′-deoxyadenosine triphosphate on in vitro RNA synthesis of plant RNA-dependent RNA polymerases

3′-deoxyadenosine triphosphate inhibited $\text{in}\text{vitro}$ [³H]UMP incorporation by RNA-dependent RNA polymerases from tobacco and cowpea plants. The inhibition of [³H]UMP incorporation could be reversed by simultaneous addition of higher ATP concentrations but not with increasing concentrations of UTP or when excess ATP was added 10 min after the inhibitor. These results suggest 3′-deoxyadenosine triphosphate competes specifically with ATP in reaction mixtures and results in premature termination of RNA synthesis $\text{in}\text{vitro}$ by RNA-dependent 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.     

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.     

Gel filtration of a complex of DNA polymerase and DNA precursor-synthesizing enzymes from a human lymphoblastoid cell line

A multienzyme complex containing at least DNA polymerase (EC 2.7.7.7), thymidine kinase (EC 2.7.1.21), dTMP kinase (EC 2.7.4.9) nucleoside diphosphokinase (EC 2.7.4.6) and thymidylate synthetase was separated from the corresponding free enzymes of DNA precursor synthesis by gel filtration of a gently lysed preparation of HPB-ALL cells (a human lymphoblastoid cell line). The isolated incorporated the distal DNA precursors [3H]thymidine or [3H]dTMP into an added DNA template at rates comparable to those observed using the immediate precursor [3H]dTTP. Measurement of the apparent overall concentrations of [3H]dTTP produced during incorporation of [3H]thymidine and of [3H]dTMP were so low as to suggest that these precursors were channelled into DNA by the operation of a kinetically linked complex of precursor-synthesizing enzymes and of DNA polymerase. The DNA polymerase inhibitor 1-beta-D-arabinofuranosylcytosine triphosphate reduced incorporation of distal precursors into DNA. However [3H]dTTP did not accumulate in the reaction mixture. This suggested that the DNA polymerase regulated the flow of substrates through the complex. The results in this paper constitute direct evidence for the existence of multienzyme complexes of DNA synthesis in mammalian cells.     

Pleiotropic effect of anticapsin on HeLa S3 cells

Anticapsin, the terminal epoxyaminoacid moiety of tetaine, inhibits irreversibly growth of HeLa S3 cells. The antibiotic decreases to a similar extent incorporation of 3H-labelled precursors into nucleic acids and protein in intact cells: inhibition of protein synthesis prevails on prolonged incubation. Also incorporation of [3H]dTTP and [3H]UTP is inhibited in the presence of anticapsin into permeabilized cells. These effects, however, are not due to the interference with DNA or RNA polymerases since anticapsin only slightly suppresses RNA polymerase activity and has no effect on DNA polymerase in the cell-free systems. The results indicate that the mechanism of antiproliferative action of anticapsin in HeLa S3 cells differs from that of tetaine and imply that inhibition of protein synthesis might be the primary effect of anticapsin.     

Effects of adenosine 3':5'-monophosphate and related agents on ribonucleic acid synthesis and morphological differentiation in mouse neuroblastoma cells in culture.

A variety of compounds were assessed for their ability to induce morphological differentiation and to affect the synthesis of RNA in uncloned mouse neuroblastoma cells in culture. The stimulation of morphological differentiation in uncloned cells after exposure for 48 hours to concentrations of 3 times 10-7 to 3 times 10-4 M papavarine or 10-9 to 10-3 M dibutyryl adenosine 3':5'-monophosphate (dibutyryl-cAMP) was associated, in part, with a concentration-dependent decrease in incorporation of [5-3H]uridine into ribosomal RNA (rRNA) and heterogeneous RNA (HnRNA). The latter effect on cellular RNA produced by papavarine occurred within 1 hour after its addition to the medium and was associated with impaired uptake of radioactive precursor into uridine nucleotides and reduction in the intracellular concentration of uridine 5'-triphosphate (UTP). Dibutytyl-cAMP produced a decreased in the specific radioactivity of UTP without affecting the concentration of UTP in the tumor cells. The effects of papavarine and dibutyryl-cAMP could be distinguished further by the 50% reduction of acetylcholinesterase activity produced by papavarine, but not by dibutyryl-cAMP. Papavarine did not, however, reduce the cellular level of the soluble enzyme, adenine phosphoribosyltransferase. Sodium butyrate, while producing morphological effects similar to those of papavarine and dibutyryl-cAMP at equimolar concentrations, caused no significant changes in the incorporation of [5-3H]uridine into rRNA and HnRNA; however, acetylcholinesterase activity was stimulated 6- to 7-fold above control levels. In contrast to the other differentiating agents examined, addition of 10-9 to 3 times 10-4 M concentrations of cAMP to the tissue culture medium enhanced morphological differentiation of nueroblastoma cells, and caused a 10- to 20-fold stimulation of the incorporation of [5-3H]uridine into rRNA and HnRNA at concentrations of 10-4 M and higher. This effect observed only at high concentrations of cyclic nucleotide was accompanied by an elevation in the specific acitivty of UTP, These studies suggest that the morphological response of neuroblastoma cells is not necessarily associated with concomitant alterations in the synthesis of RNA with agents other than cAMP. Observed changes in incorporation of [5-3H]uridine into RNA appear in most instances to be due to alterations in the uptake of uridine, and in the pool size and specific radioactivity of UTP.     

Incorporation of exogenous precursors into uridine and ribonucleic acid. Nucleotide compartmentation in the renal cortex in vivo

The possibility of compartmentation of UTP in vivo was investigated in the renal cortex of unanaesthetized rats. In addition, liver and spleen were studied in order to compare tissues with different utilization of precursors for pyrimidine nucleotide synthesis. After continuous 2h infusions of [(3)H]uridine or [(3)H]orotate, their incorporation into UTP, UDP-sugars and RNA was quantified. Rates of RNA synthesis were calculated by dividing the incorporation of precursor into RNA by the average specific radioactivity of the UTP pool. Although similar RNA-synthesis rates might have been expected with the two precursors, higher rates were found with uridine than with orotate. The relative incorporation into UDP-sugars of these precursors was also different. Similar results were obtained in the liver. In the spleen, equal amounts of both precursors were incorporated into UTP, but [(3)H]orotate incorporation did not lead to labelling of RNA. To evaluate the heterogeneity of cells with respect to the metabolism of pyrimidines, precursor incorporation was studied in isolated glomeruli and by radioautography. Incorporation into glomeruli was qualitatively similar to but quantitatively different from results in the renal cortex. Although there is obvious tissue heterogeneity, compartmentation of UTP pools is the most credible explanation for the results obtained with the renal cortex and liver. Consequently RNA and UDP-sugars may originate from two different UTP pools. Tissue heterogeneity is the likely explanation for the results obtained in the spleen. Studies of synthesis of pyrimidine and RNA, particularly in relation to growth and regeneration, must take into consideration the precursor used, the apparent existence of UTP compartmentation and the degree of cellular heterogeneity.     

Inhibitory effects of orotate on precursor incorporation into nucleic acids.

The influence of orotic acid on the incorporation of precursors into nucleic acids was studied in mice and rats and in isolated cells. In vivo, orotate levels were modified by two diets which are known to increase the rate of pyrimidine nucleotide synthesis in rat liver. Of these diets, a 1% orotate diet had greater inhibitory effects than an arginine-deficient diet on the incorporation of [3H]orotate into RNA of mouse kidney than mouse liver. This contrasted with the situation in the rat where there was a greater effect in the liver than the kidney. The situation in the rat was more readily interpreted than in the mouse in terms of previously established effects of these diets on ribonucleotide pool sizes. However, studies using [3H]adenosine as a precursor for incorporation into RNA suggested that even in the mouse the effects of orotate were on pool sizes rather than an inhibitory effect on RNA synthesis. The incorporation of [3H]thymidine into DNA was inhibited by orotate to a similar degree in cultured HTC hepatoma cells and a line of rat liver epithelial cells. An effect on DNA synthesis rather than solely on pool sizes was suggested by the observation that the pool size of dTTP was not increased by 5 mM orotate under conditions in which there was a four-fold increase in the level of UTP in HTC cells. An inhibitory effect of orotate on DNA synthesis was further supported by an observation of decreased incorporation of [3H]deoxyadenosine into DNA and a lower rate of cellular proliferation.     

Errors from using 3H-labeled ribonucleoside triphosphates to monitor nuclear RNA synthesis in vitro

The [3H]XTPs are used widely to monitor RNA synthesis in vitro. Recently, we discovered that they reflected only 40-45% of the true rate of nuclear RNA synthesis. Thus, when [8-14C]GTP was used, 1466 pmol [8-14C]GMP was incorporated per mg DNA/10 min. On the other hand, when [8-3H]GTP was used, only 564 pmol [8-3H]GMP was incorporated per mg DNA/10 min. There are three obvious factors that could have contributed to this greater than 2-fold difference in the apparent incorporation rate: commercial [8-3H]GTP sample was contaminated with substances causing the assay medium to be less efficient in RNA synthesis; 3H exchange occurred during acid washing of the [3H]RNA; and there was a greater quenching effect on [3H]RNA. Experiments were designed to test each of these alternatives. We are able to conclude that none of the above three are contributing factors. Our data also show that the 3H label was removed after it was incorporated into RNA. Similar differences were observed when 3H and 14C labeled pairs of ATP, UTP and CTP were compared. Furthermore, when nuclei were fractionated into nucleolar and nucleoplasmic fractions and carried out RNA synthesis, the loss of 3H label was observed mainly from the nucleoplasmic fraction.