RNA chain elongation on a chromatin template.

The rate of RNA chain elongation has been measured with DNA and chromatin as template. RNA propagation on chromatin is about 50% of the rate found with DNA. Kinetic experiments demonstrate that the inhibition is not due to interference with the addition of the nucleoside triphosphates. Analysis of the dependence of propagation on the Tm of DNA shows that the chromatin proteins interfere with the translocation of the RNA polymerase along the DNA template.     

Growth hormone and drug metabolism. Acute effects on nuclear ribonucleic acid polymerase activity and chromatin.

Adult male rats, subjected either to sham operation or to hypophysectomy and adrenalectomy were maintained for 10 days before treatment with growth hormone. Results of the acute effects of growth hormone on the rat liver nuclear RNA polymerase I (nucleolar) and II (nucleoplasmic) activities as well as the chromatin template capacity were then studied and compared with the growth-hormone effects on the drug metabolism described in the preceding paper (Wilson & Spelsberg, 1976). 2. Conditions for isolation and storage of nuclei for maintenance of optimal polymerase activities are described. It is verified that the assays for polymerase activities require a DNA template, all four nucleoside triphosphates, and a bivalent cation, and that the acid-insoluble radioactive product represents RNA. Proof is presented that under high-salt conditions DNA-like RNA (polymerase II) is synthesized, and that under low-salt conditions in the presence of alpha-amanitin, rRNA (polymerase I) is synthesized. 3. In the livers of hypophysectomized/adrenalectomized rats, growth hormone increases the activity of both RNA polymerase enzymes and the chromatin template capacity within 1h after treatment. The effects last for 12h in the case of polymerase II but for only 6h in the case of polymerase I. Sham-operated rats respond to growth hormone in a manner somewhat similar to that shown by hypophysectomized/adrenalectomized rats. These results, which demonstrate an enhancement of RNA polymerase I activity in response to growth hormone, support those from other laboratories. 4. Growth-hormone enhancement of the chromatin template capacity in the liver of hypophysectomized/adrenalectomized rats contrasts with previous reports. The growth-hormone-induced de-repression of the chromatin DNA could represent the basis of the growth-hormone-induced enhancement of RNA polymerase II activity in the hypophysectomized/adrenalectomized rats, although some effect of growth-hormone on the polymerase enzymes is still suggested.     

Release of template restriction in chromatin by nuclear 4.5s RNA.

The stimulatory mechanism of RNA synthesis of calf-thymus chromatin by nuclear 4.5 S RNA from the homologous tissue was investigated by using exogenously added Escherichia coli RNA polymerase. The RNA synthesis was initiated at low concentration of salt, and then the chain elongation was achieved at high concentration of ammonium sulfate in the presence of polyvinyl sulfate. Under these conditions the number of binding sites of RNA polymerase on chromatin which were capable of initiating RNA chain was increased by the addition of the 4.5 S RNA. This stimulation was presumed to result from the release of template restriction in chromatin. The polyvinyl salt minimized ribonuclease activity without changing the RNA polymerase activity bound to the template. Neither rearrangement nor release of chromatin proteins affected the amount or size of RNA produced. Preliminary analysis suggested that the molecular species of RNA produced upon the addition of the 4.5 S RNA from various tissues seemed to be heterologous.     

Hepatitis C virus NS5B polymerase exhibits distinct nucleotide requirements for initiation and elongation

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3'-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (K(app)(m) approximately 100-400 mum) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 10(3)- to 10(4)-fold, revealing a much reduced nucleotide requirement for elongation (K(app)(m) approximately 0.03-0.09 microm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 microm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3'-end of the HCV(-)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.     

Mechanism of inhibition of RNA polymerase II and poly(adenylic acid) polymerase by the O-n-octyloxime of 3-formylrifamycin SV.

Factors affecting the inhibition of RNA polymerase II from rat liver by the O-n-octyloxime of 3-formylrifamycin SV (AF/013) were investigated. Using either native or denatured calf-thymus DNA as template, almost complete inhibition of RNA polymerase II was observed when AF/013 was added directly to the enzyme. Considerable resistance to AF/013 was observed when RNA polymerase II was preincubated with denatured DNA at either 0 or 37 degrees. However, under similar conditions, no resistance was observed when enzyme was preincubated with native DNA. Only when AF/013 was added to the ongoing reaction using native DNA did a resistance to AF/013 occur. The inhibition of RNA polymerase II by AF/013 was competitive with respect to all four nucleoside triphosphate substrates. The inhibition by AF/013 remaining after enzyme-DNA complex formation also appeared competitive with nucleoside triphosphate levels. The effect of exogenous protein (bovine serum albumin, BSA) on the inhibition of RNA polymerase II was also investigated. BSA reduced the extent of inhibition by AF/013, but did not alter the competitive nature of inhibition. Concurrently, the inhibition of highly purified nuclear poly(A) polymerase from rat liver, a template independent enzyme which incorporates AMP in a chain elongation reaction, was examined. As in the case of RNA polymerase, poly(A) polymerase was inhibited by AF/013 in a manner competitive with the nucleoside triphosphate substrate. The competitive nature of inhibition of RNA polymerase by AF/013 with respect to all four nucleoside triphosphate substrates, before and after enzyme-DNA complex formation, as well as the competitive nature of inhibition of poly(A) polymerase with respect to ATP tend to indicate that the major effect of AF/013 on RNA polymerase II is at the level of the substrate binding as opposed to a specific inhibition of initiation.     

In vitro synthesis of RNA with “aggregate” enzyme,chromatin, and DNA from liver of methylazoxymethanol acetate-treated rats

“Aggregate” enzyme, chromatin and DNA preparations were isolated from livers of rats treated with the carcinogen, methylazoxymethanol (MAM) acetate. DNA template activity for RNA synthesis in vitro was unimpaired while the template activity of chromatin was slightly reduced. There was a marked inhibition of UTP incorporation into RNA, however, when the “aggregate” enzyme preparation was the source of both template and RNA polymerase. Circular dichroism analysis of the “aggregate” enzyme preparation indicated a change in conformation of the protein component. The results suggest that MAM acetate interacts with nuclear proteins and produces conformational changes which result in a decreased RNA synthesis.     

Sub-nuclear fractionation. II. Intranuclear compartmentation of transcription in vivo and in vitro

DNA-dependent RNA polymerase activities were measured in subnuclear fractions obtained from rat liver by the procedure described in the preceding paper [14]. Most of the total nuclear enzyme was recovered in a form bound to chromatin with only small amounts as free enzyme in the nucleoplasm. The multiple eukaryotic RNA polymerases were resolved according to the endogenous template to which they were bound and which they continue to transcribe in vitro. The A and B forms of the enzyme were distinguished from each other by their differential sensitivities to α-amanitin, exogenous native and denatured DNA, thermal denaturation at 45 °, Mg²⁺ and Mn² ions, high ionic strength and by the binding of ${}^{14}\text{C-methyl}\text{-γ-}\text{amanitin}$. RNA polymerase B (α-amanitin-sensitive) was exclusively recovered in the nucleoplasmic and euchromatin fractions. RNA polymerase A was recovered in the dispersed nucleolar as well as in heterochromatin. By assaying in the presence of α-amanitin subnuclear fractions that had been pre-incubated at 45 °C a third enzyme (form C) was located exclusively in heterochromatin fractions. Only the euchromatin associated RNA polymerase B was capable of initiating the synthesis of new RNA chains in vitro on endogenous template at low ionic strength. Raising the ionic strength abolished initiation but accelerated chain elongation by this form of enzyme.When nuclear RNA was labelled in vivo, newly made RNA turned over rapidly in the nucleoplasm but accumulated in the euchromatin + membrane fraction. RNA in the nucleolar fraction accumulated gradually after a lag period, whereas a significant amount of rapidly-labelled nuclear RNA was recovered in the heterochromatin fractions. The distribution of RNA labelled in vivo compared with that of RNA polymerase activities suggested that RNA synthesized in vivo is rapidly translocated from its site of synthesis to some other sites within the nucleus.     

Modification of the template capacity of liver chromatin for form-B ribonucleic acid polymerase by food intake in rats under controlled feeding schedules.

Nuclei from liver of rats accustomed to eating during the first 8h of a daily 12h dark period demonstrate an increased capacity to synthesize RNA 6H after the beginning of the feeding period. 2. This increase is accompanied by a higher yield of extractable form-B DNA-dependent RNA polymerase activity. 3. The endogenous RNA polymerase activity associated with nuclear chromatin is also stimulated by food intake. Both purified and chromatin-associated form-B enzyme activities exhibit different ionic strength requirements after food intake. 4. The sensitivity of exogenous (added) form-B-enzyme to changes in ionic strength changes after feeding when chromatin is used as template. 5. Chromatin extracted from the liver of fed rats is a better template for form-B-enzyme than chromatin extracted from starved rats.