Utilizations of various uridine 5'-triphosphate analogues by DNA-dependent RNA polymerases I and II purified from liver nuclei of the cherry salmon (Oncorhynchus masou)

Various 5-substituted UTPs (methyl, ethyl, n-propyl, n-butyl, fluoro, chloro, bromo, and iodo) and sulfur-containing UTP analogues (4-thio-, 2-thio-, 5-methyl-2-thio-, and 5-methyl-4-thio-) were synthesized chemically and their utilization by DNA-dependent-RNA polymerases I and II of the cherry salmon (Oncorhynchus masou) were studied in substitution experiments under the condition of limited RNA synthesis in vitro. RNA polymerase I utilized the 5-methyl-, chloro, bromo, and iodo derivatives of UTP more efficiently than unmodified UTP, but RNA polymerase II utilized UTP most efficiently. 5-Methyl-4-thiouridine 5'-triphosphate (4-thio TTP) was utilized more efficiently than UTP by RNA polymerase I. On the other hand, it was found that 4-thio TTP was a selective substrate for RNA polymerase I and that its incorporation by RNA polymerase II was very slow. Thus recognition of UTP analogues as substrates by RNA polymerase I and II was different. These observation were attributed from kinetic analyses to differences in catalytic activity (Vmax).     

Viral RNA synthesis and levels of DNA-dependent RNA polymerases during replication of adenovirus 2.

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin alpha-amanitin was used to determine the relative and absolute levels of RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication of from replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

Viral RNA Synthesis and Levels of DNA-Dependent RNA Polymerases During Replication of Adenovirus 2

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin α-amanitin was used to determine the relative and absolute levels of RNA synthesis by RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was monitored by hybridization to viral DNA, and of viral 5.5S RNA, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

EARLY EVENTS IN LYMPHOCYTE TRANSFORMATION BY PHYTOHEMAGGLUTININ : I. DNA-Dependent RNA Polymerase Activities in Isolated Lymphocyte Nuclei

The DNA-dependent RNA polymerase activities of isolated nuclei from lymphocytes were examined after stimulation with phytohemagglutinin (PHA). The nuclear fraction was prepared with Mg⁺⁺ or Mn⁺⁺ to distinguish between polymerase I (nucleolar) and polymerase II (nucleoplasmic). Distinction between polymerases II and III was obtained by the addition of α-amanitin to the reaction mixture. The results indicated that within 15 min after exposure to PHA the activity of polymerase I increased. Polymerase II activity increased after 1 hr. The enhancement was linear for 6 hr and then leveled off for the subsequent 48 hr. Small increase in polymerase III activity was observed at 48 hr. Inhibition of protein synthesis at the time of exposure to PHA did not prevent the increase in activities during the initial 6 hr. These results imply that the initial increase in enzymatic activities is dependent upon preexisting polymerase molecules and/or factors.     

Ribonucleic acid labelling and nucleotide pools during compensatory renal hypertrophy

During the first 48h of compensatory renal hypertrophy induced by unilateral nephrectomy, RNA content per cell increased by 20-40%. During this period, rates of RNA synthesis derived from the rates of labelling of UTP and RNA after a single injection of [5-(3)H]uridine showed no change in the rate of RNA synthesis (3.1nmol of UTP incorporated into RNA/min per mg of RNA). ATP and ADP pools were not changed. The rate of RNA synthesis was considerably in excess of the increment of total RNA appearing in the kidneys. With [5-(3)H]uridine as label, only continuous infusion for 24h could produce an increase (60%) in the specific radioactivity of renal rRNA in mice with contralateral nephrectomies. With a single injection of [methyl-(3)H]methionine used to identify methyl groups inserted into newly synthesized rRNA, the specific radioactivity of this rRNA was unchanged 5h after contralateral nephrectomy, increased by 60% at 9-48h, and returned to normal values at 120h. Most RNA synthesized in both nephrectomized and sham-nephrectomized mice has a short half-life. Since total cellular RNA content increases in compensatory hypertrophy despite unchanged rates of rRNA synthesis, the accretion of RNA might involve conservation of ribosomal precursor RNA or a change in rate of degradation of mature rRNA.     

Differential inhibitory effects of 5-substituted 1-beta-D-xylofuranosyluracil 5'-triphosphates and related nucleotides on DNA-dependent RNA polymerases I and II from the cherry salmon (Oncorhynchus masou)

Various 5-substituted 1-beta-D-xylofuranosyluracil 5'-triphosphates (hydrogen, methyl-, ethyl-, n-propyl, n-butyl, fluoro-, chloro-, bromo-, and iodo derivatives) and some of the 3'-deoxyribofuranosyl nucleotides (3'-deoxy UTP and 3'-deoxy TTP) were synthesized chemically and their inhibitory effects on DNA-dependent RNA polymerases I and II of the cherry salmon (Oncorhynchus masou) were studied systematically. These 3'-modified UTP analogues could not be utilized as substrates in place of UTP, but they did inhibit the incorporation of UMP into RNA in vitro. In contrast, 2'-modified UTP analogues, such as 2'-dTTP and Ara TTP, were neither substrates nor inhibitors. Kinetic analysis showed that the inhibition by these compounds was essentially competitive with substrate UTP. The K1 values of RNA polymerase I for the analogues were smaller (2-6 microM) than the Km value for UTP (8 microM), but those for xylo-EtUTP, xylo-PrUTP, and xylo-BuUTP were larger (about 20 microM) than the Km for UTP. In contrast to these alkyl groups with steric and electron-donating effects, halogen groups have electron-withdrawing effects on the uracil nucleus. Therefore, it was concluded that the inhibitory activity of these analogues on RNA polymerase I was not affected by the inductive effects of substituent groups at the 5-position of uracil nucleus but by their steric effects. On the other hand, all of the K1 values of RNA polymerase II for UTP analogues were smaller (0.4-3 microM) than the Km value for UTP (4 microM). In this case, neither steric effect nor an inductive effect of substituents on UTP analogues influenced the inhibitory activity towards RNA polymerase II.     

Effects of partial hepatectomy on RNA polymerase activities in mouse liver

Chromatin-bound and poly[d(A-T)]dependent RNA polymerase I plus III and II activities of mouse liver were analysed 24 and 48 hr after partial hepatectomy. Chromatin-bound RNA polymerase I plus III activity showed an increase of 57% at 24 hr and 51% at 48 hr after partial hepatectomy. There was a decrease in chromatin-bound RNA polymerase II activity of 15% at 24 hr and 34% at 48 hr after partial hepatectomy. There was no significant changes in poly[d(A-T)]dependent RNA polymerase activities. Heparin caused an approximately 10-fold increase in chromatin-bound RNA polymerase II activity. The stimulation by heparin was significantly increased 48 h after partial hepatectomy. Anaesthesia and/or surgery had great influence on RNA polymerase activities. At 24 hr after operation, chromatin-bound RNA polymerase I plus III and II activities were depressed, and the liver cell chromatin was more susceptible to stimulation by heparin.     

Lutropin stimulation of RNA synthesis in corpus luteum chromatin.

Lutropin and human choriogonadotropin stimulated the endogenous chromatin-associated polymerase activity in purified chromatin prepared from nuclei of bovine corpus luteum. Chromatin was incubated in two different buffer systems: one that mainly supports the activity of polymerase I, another that supports the activity of polymerase II and is largely alpha-amanitin sensitive. The hormones lutropin and chorigonadotropin stimulated an increase in the rate of incorporation of [14C]ATP or [14C]UTP into RNA in both buffer systems. Follitropin, prolactin and beta-corticotropin had no stimulatory effect. Neither the alpha nor beta subunit of lutropin stimulated RNA synthesis. When premixed, the subunits rapidly formed the active molecule. A maximum response to RNA synthesis was achieved by a 10(-9) M concentration of human choriogonadotropin. Considerable activity was obtained at 10(-11) M human choriogonadotropin. There was no lutropin stimulation to RNA synthesis using calf thymus DNA and Escherichia coli RNA polymerase.     

Early effects of oestradiol-17β on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus

Oestradiol-17β (1.0μg) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10–15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1–2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17β was an increase (30–60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17β and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in `high-salt conditions' can be completely eliminated by α-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se.     

In vitro RNA synthesis in oocyte nuclei of the newt Notophthalmus

An incubation medium is described which supports RNA synthesis in isolated oocyte nuclei of the newt Notophthalmus, and which permits subsequent autoradiographic examination of the lampbrush chromosomes and nucleoli. By using different concentrations of α-amanitin we distinguish RNA synthesis due to RNA polymerases I, II and III. All RNA synthesis on loops is inhibited by 0.5 μ/ml of α-amanitin and is therefore due to polymerase II. Polymerase III is responsible for RNA synthesis at a small number of discrete sites in condensed chromatm. These include the centromere bars of three of four chromosomes, which probably represent 5S RNA synthesis, as well as 15–20 lesser sites scattered elsewhere. Polymerase I activity is confined to the nucleoli. Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

Temporal studies of factors associated with changes in transcription during Q fever

Temporal studies were made of factors associated with increased RNA synthesis in guinea pig liver during Q fever. DNA-dependent RNA polymerase activities increased immediately after infection. The major distribution of RNA polymerase classes shifted from class II to class I during infection. Ornithine decarboxylase activity was induced or stimulated soon after infection and remained elevated throughout the four-day period studied. S-Adenosylmethionine decarboxylase activity increased on the first day after infection and subsequently declined. Concomitantly elevated concentrations of the polyamines putrescine, spermidine and spermine reached a maximum on the first day after infection and then decreased. A model is presented to integrate these and other results to explain how RNA synthesis may be regulated during infection.     

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.     

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.     

Differential effects of cordycepin triphosphate and 9 beta-D-arabinofuranosyladenine triphosphate on tRNA and 5 S RNA synthesis in isolated nuclei.

Although cordycepin 5'-triphosphate (3'-dATP), at low concentrations, preferentially inhibits chromatin-associated poly(A) synthesis in isolated nuclei, higher levels of the inhibitor prevent both rRNA (RNA polymerase I activity) and hnRNA (RNA polymerase II activity) synthesis in vitro (Rose, K.M., Bell, L.E. and Jacob, S.T. (1977) Nature 267, 178-180). The present studies demonstrate that this nucleotide can also inhibit tRNA and 5 S RNA synthesis (RNA polymerase III activity). At 50-200 microgram/ml, 3'-dATP inhibits incorporation of [3H]UTP into tRNA and 5 S RNA by approximately 65%, whereas the syntheses of these RNAs were completely blocked when [3H]GTP was used as the substrate. These data suggest the formation of poly(U) in the tRNA and 5 S RNA regions, which is resistant to 3'-dATP. In contrast, another ATP analog, Ara-ATP, which selectively inhibits poly(A) synthesis, does not block tRNA and 5 S RNA synthesis in isolated nuclei. The production of these RNA species in isolated nuclei is also insensitive to Ara-CTP and 2'-dATP. These data suggest that 3'-dATP exerts general inhibitory effects on RNA synthesis and further substantiate the conclusion that Ara-ATP is a selective inhibitor of the polyadenylation reaction in vitro.