Showing papers on "RNA polymerase III published in 1975"

A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II.

Abstract: An improved method for the purification of the alpha-amanitin-sensitive deoxyribonucleic acid dependent ribonucleic acid polymerase [ribonucleosidetriphosphate:RNA-nucleotidyltransferase, EC 2.7.7.6-A1 (RNA polymerase II or RNA polymerase B) from wheat germ is presented. The method involves homogenization of wheat germ in a buffer of moderate ionic strength, precipitation of RNA polymerase with Polymin P (a polyethylenimine), elution of RNA polymerase from the Polymin P precipitate, ammonium sulfate precipitation, and chromatography on DEAE-cellulose and phosphocellulose. RNA polymerase II is purified over 4000-fold with a 60% recovery, resulting in a yield of 25-30 mg of RNA polymerase from 1 kg of starting material.

Distinct molecular structures of nuclear class I, II, and III DNA-dependent RNA polymerases.

Abstract: Class III RNA polymerases purified from the murine plasmacytoma MOPC 315 and from Xenopus laevis ovaries were compared. The subunit structures of the chromatographically distinct murine enzymes IIIA and IIIB were indistinguishable and were remarkably similar to that of the amphibian enzyme III. The plasmacytoma class III RNA polymerases were also compared with purified plasmacytoma RNA polymerases I and II. Sedimentation studies indicated that RNA polymerase III si significantly larger than RNA polymerase II, which is slightly larger than RNA polymerase I. Structural analyses showed that the molecular weights of the large subunits present in the class III enzymes (138,000 and 155,000) differ from those of the class II enzymes (140,000 and either 170,000, 205,000, or 240,000) and from those of the class I enzymes (117,000 and 195,000). Some low-molecular-weight subunits are also unique to each enzyme class. These results clearly distinguish the class I, II, and III enzymes on a structural basis. In addition, polypeptides of molecular weight 29,000 and 19,000 were found in all enzyme classes, a polypeptide of molecular weight 52,000 was found only in class I and III enzymes, and a polypeptide of molecular weight 41,000 was found only in class II and III enzymes. These findings are discussed in terms of the function and regulation of the RNA polymerases.

Partial purification and properties of calf thymus deoxyribonucleic acid dependent RNA polymerase III.

Abstract: DNA-dependent RNA polymerase III (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.-7.6) has been isolated and partially purified from calf thymus tissue. Significant amounts of enzyme III are present in this tissue (up to 15% of the total activity of thymus homogenates). This enzyme has been characterized with respect to its chromatographic properties, broad ammonium sulfate optimum (0.04-0.2 M), template requirements, divalent metal optima, and its unique alpha-amanitin sensitivity (50% inhibition of activity occurring at an alpha-amanitin concentration of 10 mug/ml).

Comparative effect of heparin on RNA synthesis of isolated rat-liver nucleoli and purified RNA polymerase A.

Abstract: The polyanion heparin has been employed to study the interaction of rat liver DNA-dependent RNA polymerase A and its template under various conditions. Heparin very efficiently inhibits polymerase molecules, which are not bound to DNA or are associated with the template in a loose, i.e., non-specific fashion. Purified nucleoli, isolated from rat liver nuclei, contain RNA polymerase A in abundant quantities of which only a portion is bound in a transcriptional complex. Excess enzyme, which is contained in the nucleolus in a quasi free form, can be transferred to an exogenously added template and can be completely inhibited by the prior addition of heparin. The enzyme contained in a transcriptional complex, however, initiated in vivo and completing these RNA chains in vitro, is fully resistant to heparin. In contrast to these results it has been found that RNA polymerase A extracted from nuclei and purified by various chromatographic steps does not form heparin-resistant complexes, even after the enzyme has been bound to the DNA template. Moreover it has been found that purified RNA polymerase A transcribes truly native DNA extremely poorly, indicating that the enzyme is highly deficient in the act of initiation on duplex DNA. It is therefore questionable whether the interaction of the purified enzyme and isolated DNA represents binding to true initiation complexes as is observed in the intact nucleolus.

DNA‐Dependent RNA Polymerase C

Abstract: DNA-dependent RNA-polymerase C, initially shown to exist in rat liver tissue and characterized by its sensitivity toward high concentrations of α-amanitin, has been shown to exist in HeLa cells and to be present in variable amounts in the nucleus of the rat liver cell. Enzyme C has been shown to co-chromatograph with nucleolar RNA polymerase A (or I) on DEAE-cellulose and can clearly be separated from the latter by chromatography on DEAE-Sephadex. It is possible that RNA polymerase C may occur in multiple forms although the significance of this finding is unclear at present. From its similar chromatographic properties and sensitivity toward α-amanitin it is possible that RNA polymerase C corresponds to polymerase III.

Sequential Stimulation of Nuclear RNA Polymerase Activities in Livers from Thyroidectomized Rats Treated with Triiodothyronine

Abstract: A single ip injection of triiodothyronine (T3; 30 µg /lOOg BW) to thyroidectomized rats markedly stimulates RNA synthesis in isolated liver nuclei. The increased level of RNA synthesized in vitro by isolated nuclei does not depend on a reduced degradation of the nascent RNA molecules, since ribonuclease activities are not affected by the administration of T3. In addition, our results have confirmed previous findings of Tata et al. that the increase in nucleolar α-amanitin-resistant RNA polymerase I activity at low ionic strength always preceded the rise of the nucleoplasmic α-amanitinsensitive RNA polymerase II activity at high ionic strength. Moreover, it has been found that a significant increase in an α-amanitin-resistant activity at high ionic strength occurs as early as 10 h after hormone injection. This enzyme, which forms RNA with a U to G ratio significantly higher than that of RNA synthesized by the nucleolar α-amanitinresistant enzyme, is probably nucleoplasmic RNA polymerase III which is though...

Effects of bacteriophage T4-induced modification of Escherichia coli RNA polymerase on gene expression in vitro.

Abstract: After T4 bacteriophage infection of E. coli a complex series of events take place in the bacterium, including gross inhibition of host transcription and discrete changes in the classes of the genes of T4 that are transcribed. Accompanying these changes in the pattern of transcription one finds T4-induced changes in the RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase). The effects of modified polymerase on transcription can be advantageously analyzed in a DNA-directed cell-free system for protein synthesis. In this system gene activity is measured indirectly by the amounts and types of proteins sythesized. In the DNA-directed cell-free system this modified polymerase, like normal polymerase, transcribes T4 DNA with a high efficiency but transcribes bacteriophage lambda and host DNA very poorly. Polymerase reconstruction experiments show that modification of the alpha subunit of the RNA polymerase is sufficient for inhibition of host transcription. Host transcription is also inhibited in vitro by T4 DNA. This latter type of inhibition is presumed to involve competition between host DNA and T4 DNA for some factor essential for transcription. The T4-modified polymerase transcribes from T4 DNA many of the same genes as normal unmodified polymerase; it also shows a capability for transcribing certain "non-early" T4 genes which is enhanced in the presence of protein-containing extracts from T4-infected cells.

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

Abstract: 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.

Low-salt and high-salt RNA polymerase activity during preimplantation development in the mouse.

Abstract: one of the two main forms of the enzyme. A situation of this kind has been found during the development of Rana pipiens embryos (Kohl, Norman & Brooks, 1973); Polymerase I (the nucleolar form) and Polymerase II (nucleoplasmic) behave differently. The levels of Form II do not change during development despite changing patterns of DNA-like RNA synthesis, while Form I increases in the nuclei after gastrulation when ribosomal RNA synthesis begins, suggesting that the expression of the ribosomal cistrons may be controlled in this case by the availability of the corresponding polymerase. We have attempted, therefore, to discriminate between the levels of Polymerases I and II at various developmental stages of the mouse embryo by measuring at two different ionic strengths the enzymatic activity present in embryo homogenates. The activities of RNA polymerases are markedly influenced by the ionic strength. In the presence of excess native DNA, Polymerase I shows optimal activity at 25 to 50 mM-ammonium sulphate, Polymerase II at 100 to 120 min (Gissinger, Kedinger & Chambon, 1974). The technical procedures used were those previously reported (Siracusa, 1973), with the following main differences. Native DNA was used as template in the assay; tritiated uridine triphosphate ([3H]UTP) concentration was raised to 50 μ (NEN, sp. act. 19-8 Ci/mmol), incubation time was prolonged to 90 min. The final incubation mixture (40 μ ) contained 50 mM-tris-HCl, pH 7-9; 2-5 mM-MgCl2 ; 2 mM-MnCl2 ; 0-2 mM-ATP; 0-2 mM-GTP; 0-2 mM-CTP; 50 ^m-[3H]UTP; 0-2 mg native DNA/ml (calf thymus); 12-5% glycerol; 0-05 mM-EDTA; 0-5 mM-dithiothreitol; 2-5 mmNaF; 0-5 mg BSA/ml; 25 or 125 mM-(NH4)2S04. A total of 4140 embryos was used.

Structure, function, and regulation of rna polymerases in animal cells

Abstract: . The mouse plasmacytoma class I, II, and III RNA polymerases have been purified and shown to have distinct structures and functions. The two large molecular weight subunits and some low molecular weight subunits differ between the class I, II, and III enzymes. However, some small subunits appear common to two or to three enzyme classes. α-Amanitin was used to distinguish the endogenous RNA polymerase activities of the class I, II, and III RNA polymerases in isolated nuclei and to show their functions, respectively, in transcription of the genes for the rRNAs, the HnRNAs, and the transfer and 5S RNAs. The cellular activity levels of solubilized RNA polymerases I and III vary with the physiological state of the cell in different mouse tissues, suggesting that the activities of the genes which they transcribe are regulated in part by specific enzyme levels in adult cell types. The increased RNA polymerase I levels in malignant mouse plasmacytoma cells reflect primarily increased enzyme concentrations. The levels of RNA polymerase II in mouse tissues show much less variability suggesting similar rates of HnRNA synthesis in these cell types or cellular excesses of this enzyme. During very early embryonic development in X. laevis all RNA polymerases are present in vast cellular excess and changes in specific gene function are not accompanied by changes in total enzyme levels, suggesting that factors other than enzyme levels are involved in regulating the transcription of all genes during this developmental period.

A comparative study of DNA-dependent RNA polymerases from rat ascites hepatoma cell nuclei and from rat liver nuclei.

Abstract: DNA-dependent RNA polymerases (EC 2.7.7.6) were extracted and partially purified form the nuclei of rat ascites hepatoma cells (AH-130) induced by 4-dimethylaminoazobenzene. The patterns of RNA synthesis and the properties of these enzymes were compared with enzymes from the nuclei of rat liver. The specific activity of RNA polymerase in the homogenate from the nuclei of AH-130 cells was the same as normal rat liver nuclei. RNA polymerase was solubilized from the homogenate at high ionic strength and separated into two forms by DEAE-Sephadex column chromatography. Enzymatic characterization showed that these enzymes corresponded to RNA polymerase I and II. RNA polymerase I more effectively transcribed native DNA than denatured DNA at low salt concentration, but at high salt concentration RNA polymerase I effectively transcribed denatured DNA. RNA polymerase II more effectively transcribed denatured DNA. In AH-130 cells the activity of RNA polymerase I was 4 to 5 times higher than RNA polymerase II, and in rat liver the activity of RNA polymerase I was 1.5 to 2 times higher than RNA polymerase II. The activity of RNA polymerase I in AH-130 cells may have increased by induction.