Adrenocorticotropic hormone stimulation of adrenal RNA polymerase I and III activities. Nucleotide incorporation into internal positions and 3' chain termini.

In the presence of 50 mM (NH4)2SO4 and low concentrations of alpha-amanitin (7.7 mug/ml), adrenal nuclei synthesize predominately rRNA as characterized by size and base composition. Approximately 10% of the RNA synthesized under these conditions sediments at 4-5 S; this RNA synthesizing activity is inhibited by high concentrations of alpha-amanitin (231 mug/ml) indicating the presence of RNA polymerase III activity. ACTH administration to guinea pigs results in a twofold increase in adrenal nuclear RNA polymerase I and III activities at 14 hr of hormone treatment. Analysis of the amount of radiolabeled nucleoside triphosphate incorporated in vitro into 3' chain termini and into internal nucleotide positions has been utilized to measure the number of RNA chains and the average chain length synthesized in vitro. Incorporation into 3' chain termini is not changed by ACTH; incorporation into internal nucleotides is doubled in parallel with the increase in RNA polymerase I activity. These results are not due to an altered Km of RNA polymerase I for the four nucleoside triphosphates, nor to differential R Nase or phosphatase activity. These studies suggest that the regulation of RNA polymerase I by ACTH is accomplished in part through an increase in the rate of RNA chain elongation.     

Inhibition of cellular DNA polymerase alpha and human cytomegalovirus-induced DNA polymerase by the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine.

The triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine were examined for their inhibitory effect on highly purified cellular DNA polymerase alpha and human cytomegalovirus (Towne strain)-induced DNA polymerase. These two nucleoside triphosphates competitively inhibited the incorporation of dGMP into DNA catalyzed by the DNA polymerases. The virus-induced DNA polymerase had greater binding affinity for the triphosphate of 9-(2-hydroxyethoxymethyl)guanine (Ki, 8 nM) than for the triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (Ki, 22 nM), although the nucleoside of the latter compound was strikingly more effective against human cytomegalovirus replication in cell cultures than the nucleoside of the former. The Ki values of these two nucleoside triphosphates for alpha polymerase were 96 and 146 nM, respectively, and were 7- to 12-fold higher than those for the virus-induced enzyme. These data indicated that virus-induced DNA polymerase was more sensitive to inhibition by these two nucleoside triphosphates than was the cellular alpha enzyme.     

Occurence of nucleoside polyphosphates in rapidly labelled RNA preparations from radish seedlings (Raphanus sativus.)

Summary Preparations of rapidly labelled RNA are usually heavily contaminated by rapidly labelled low molecular weight components. This contaminating fraction mainly contains nucleoside triphosphates, as previously reported, but also nucleoside tetra, penta and hexaphosphates. These highly phosphorylated nucleosides are, according to recent literature, likely candidates for an important regulatory role in the coordination of a variety of biochemical reactions. This is the first demonstration of their occurence in higher plants and a method is presented to separate them from other usual nucleotides.Abbreviation PEI poly-ethylene-imine     

Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polymerase

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.     

Regulation of RNA synthesis in higher plant cells by the action of a nucleoside triphosphatase

The influence of nucleoside triphosphates in relation to divalent cations on RNA synthesis of cells from a suspension culture from parsley was investigated. The data obtained from experiments with isolated nuclei and with an in vitro system with highly purified RNA polymerase I were compared with a chromatin-bound nucleoside triphosphatase activity within the nucleus. The results might suggest a regulatory role of the nucleoside triphosphatase activity in RNA synthesis.Abbreviations NTP nucleoside triphosphates - NTPase nucleoside triphosphatase     

Protein P4 of the bacteriophage phi 6 procapsid has a nucleoside triphosphate-binding site with associated nucleoside triphosphate phosphohydrolase activity.

Bacteriophage phi 6 contains three segments of double-stranded RNA. The procapsid consists of proteins P1, P2, P4, and P7, which are encoded by the viral L segment. cDNA copies of this segment have been cloned into plasmids that direct the production of these proteins, which assemble into polyhedral procapsids. These procapsids are capable of packaging plus-sense phi 6 RNA in the presence of nucleoside triphosphate and synthesizing the complementary minus strand to form double-stranded RNA. In this article, we report the presence of a nucleotide-binding site in protein P4. The viral procapsid and nucleocapsid exhibit a nucleoside triphosphate phosphohydrolase activity that converts nucleoside triphosphates into nucleoside diphosphates.     

The synthesis of 2'-methylseleno adenosine and guanosine 5'-triphosphates

Modified nucleoside triphosphates (NTPs) represent powerful building blocks to generate nucleic acids with novel properties by enzymatic synthesis. We have recently demonstrated the access to 2'-SeCH(3)-uridine and 2'-SeCH(3)-cytidine derivatized RNAs for applications in RNA crystallography, using the corresponding nucleoside triphosphates and distinct mutants of T7 RNA polymerase. In the present note, we introduce the chemical synthesis of the novel 2'-methylseleno-2'-deoxyadenosine and -guanosine 5'-triphosphates (2'-SeCH(3)-ATP and 2'-SeCH(3)-GTP) that represent further candidates for the enzymatic RNA synthesis with engineered RNA polymerases.     

Kinetics of the interaction of methylene diphosphonic acid and inorganic pyrophosphate with DNA-dependent RNA-polymerase from calf thymus

The kinetics of interaction of PPi and its diphosphonic analog, methylenediphosphonic acid (MDPA), with nucleoside triphosphates, DNA and Mg2+ binding sites of DNA-dependent RNA polymerase II from calf thymus was investigated. The values of apparent Km in the NTP polymerization reaction for ATP and CTP equal to 2.7 X 10(-4) and 1.8 X 10(-4) M, respectively, were determined. It was shown that MDPA and PPi competitively inhibited the RNA polymerase reaction with respect to nucleoside triphosphate. The inhibition constants (Ki) of ATP and CTP incorporation for MDPA were 2.2 X 10(-4) and 3.3 X 10(-4) M, respectively, while those of the nucleoside triphosphate incorporation for PPi were equal to 1.4 X 10(-4) and 2.0 X 10(-4) M, respectively. MDPA and PPi were incompetitive inhibitors of template (DNA) and Mn2+. A possible mechanism of inhibition of the RNA polymerase reaction by MDPA is proposed.     

Structure-activity relationship of uridine-based nucleoside phosphoramidate prodrugs for inhibition of dengue virus RNA-dependent RNA polymerase

To identify a potent and selective nucleoside inhibitor of dengue virus RNA-dependent RNA polymerase, a series of 2′- and/or 4′-ribose sugar modified uridine nucleoside phosphoramidate prodrugs and their corresponding triphosphates were synthesized and evaluated. Replacement of 2′-OH with 2′-F led to be a poor substrate for both dengue virus and human mitochondrial RNA polymerases. Instead of 2′-fluorination, the introduction of fluorine at the ribose 4′-position was found not to affect the inhibition of the dengue virus polymerase with a reduction in uptake by mitochondrial RNA polymerase. 2′-C-ethynyl-4′-F-uridine phosphoramidate prodrug displayed potent anti-dengue virus activity in the primary human peripheral blood mononuclear cell-based assay with no significant cytotoxicity in human hepatocellular liver carcinoma cell lines and no mitochondrial toxicity in the cell-based assay using human prostate cancer cell lines.     

Specific labelling of the active site of T7 RNA polymerase.

We describe a method for specifically labelling T7 RNA polymerase at (or near) the active site. Enzyme molecules that have been modified by covalent attachment of a benzaldehyde nucleotide derivative in the presence of template DNA are subsequently incubated with radioactively labelled nucleoside triphosphates. Labelling of the enzyme occurs as a result of the formation of the first phosphodiester bond. The labelling is template-directed and the expected specificity of initiation at individual T7 promoters is observed. The label has been localized to an 80 kd tryptic fragment that contains the carboxy-terminal portion of the enzyme.     

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.     

NUCLEAR-CYTOSOL INTERACTIONS THAT FACILITATE RELEASE OF RNA FROM MOUSE BRAIN NUCLEI

Abstract— Mouse brain nuclei were incubated in vitro under conditions that primarily lead to the synthesis of radioactive polydisperse and messengerlike nuclear RNA. After incubation the effects of Mg² concentrations, nucleoside triphosphate levels and brain cytosol were examined with regard to their ability to influence the release of RNA from brain nuclei. The presence of 8 mM -MgCl₂ and a total of 0.3 mM-nuclcoside triphosphates during the labelling procedure allowed only a minimal amount of RNA to be released. However, when the MgCl₂ was decreased to 2 mM and the nucleoside triphosphates were increased to 1 mM, a stimulation of RNA release was observed. The addition of unfractionated brain cytosol under these conditions resulted in an inhibition of RNA release.
G-100 Sephadex filtration removed detectable RNase activity from the cytosol preparations and allowed the identification of fractions that were able to facilitate nuclear RNA release by 3-fold. The fractions that stimulated release did not have detectable levels of RNase, protease or DNA-dependenl RNA polymerase. Under conditions that provided maximum nuclear RNA release by both labelled mouse brain and neuroblastoma nuclei, no release of DNA could be measured. The cytosol fractions that facilitated RNA release did not have a high affinity for nuclear RNA or an ability to stimulate nuclear RNA synthesis. However, other components in the cytosol were shown to stimulate RNA metabolism in isolated mouse brain nuclei and to have a relatively high binding affinity to nuclear RNA. Further purification of the RNA release components in the brain cytosol by DEAF. Sephadex chromatography allowed an increase in specific activity of at least 40-fold. The thermal lability, effective filtration size, and solubility in phenol suggested that the cytosol factors that facilitiated nuclear RNA release were associated with cellular proteins.     

The effect of low substrate concentrations on the extent of productive RNA chain initiation from T7 promoters A1 and A2 by Escherichia coli RNA polymerase

The extent of productive RNA chain initiation in vitro by Escherichia coli RNA polymerase holoenzyme from the bacteriophage T7 A1 and A2 promoters on purified T7 DNA templates has been investigated at very low concentrations of the ribonucleoside triphosphate substrates. As the concentration of ribonucleoside triphosphates in the reaction is decreased from 10 to 1 micro M, the extent of productive RNA chain initiation at these promoter sites drops precipitously at about 3 micro M. At 1 micro M substrate concentration, productive chain initiation from the A1 promoter does not occur even after extended incubation although the dinucleoside tetraphosphate pppApU is produced at a significant rate under these conditions. The reason for the inability of RNA polymerase to carry out productive RNA chain initiation at 1 micro M substrate concentration is not yet understood. The phenomenon is not due to substrate consumption, enzyme inactivation, or a requirement for a nucleoside triphosphatase activity in the reaction. The possibility is raised that there are additional nucleoside triphosphate binding sites on E. coli RNA polymerase which play some role in the process of productive RNA chain initiation.     

RNA polymerase I from higher plants. Evidence for allosteric regulation and interaction with a nuclear phosphatase activity controlled NTP pool.

RNA polymerase I was isolated from parsley cells grown in suspension culture and from soybean hypocotyls. Kinetic studies of the enzyme revealed that RNA polymerase I is an allosteric regulated enzyme. The enzyme activity was influenced by nucleoside triphosphates (NTP) and divalent cations. NTP exceeding a 1:1 ratio of these two components acted as allosteric inhibitors, contrary to free divalent cations, which had promotive effects on the RNA polymerase I. Furthermore, isolated nuclei from parsley exhibited a powerful nucleoside triphosphatase (NTPase) activity. Contrary to RNA polymerase I, this enzyme was stimulated by NTP exceeding the 1:1 ratio of NTP and divalent cations. Free divalent cations had an inhibitory effect. Assuming that a causal connection of these two processes does exist, a possible role of this NTPase would be the control of NTP pools in relation to divalent cations and thus regulating RNA synthesis.