Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase III from the mouse plasmacytoma, MOPC 315.

Class III DNA-dependent RNA polymerases were purified from the mouse plasmacytoma, MOPC 315. RNA polymerases IIIA and IIIB were solubilized from a whole cell extract and resolved by chromatography on DEAE-Sephadex. Chromatography on DEAE-cellulose, DEAE-Sephadex, CM-Sephadex, and phosphocellulose ion exchange resins and sedimentation in sucrose density gradients yielded chromatographically homogeneous Enzymes IIIA and IIIB which were purified approximately 22,000 and 53,000-fold respectively, relative to whole cell extracts. The specific activity of these enzymes was comparable to that reported for other purified eukaryotic RNA polymerases. Sucrose gradient sedimentation analysis suggested a molecular weight of approximately 650,000 for each of the class III enzymes.     

The sensitivity of RNA polymerases I and II from Novikoff hepatoma (N1S1) cells to 3''-deoxyadenosine 5''-triphosphate.

The synthesis of ribosomal precursor RNA in Novikoff hepatoma (N1S1) cells is very sensitive to cordycepin (3'-dA). The synthesis of hnRNA, however, is resistant to inhibition concentrations of 3'-dA that completely block the synthesis of 45S ribosomal RNA precursor. We have examined the RNA polymerases present in these cultured cells with regard to their sensitivity to cordycepin 5'-triphosphate (3'-dATP) in an effort to explain the differential inhibition of RNA synthesis observed in vivo. RNA polymerases I and II were characterized on the basis of their chromatographic behavior on DEAE-Sephadex, as well as the response of their enzymatic activities to ionic strength, the divalent metal ions Mn2+ and Mg2+, and the toxin alpha-amanitin. For both enzymes the inhibition of in vitro RNA synthesis by 3'-dATP was competitive for ATP. The km values for ATP and the K1 values for 3'-dATP for the two enzymes were quite similar. RNA polymerase II, the enzyme presumed responsible for hnRNA synthesis, was actually slightly more sensitive to 3'-dATP than RNA polymerase I, the enzyme presumed responsible for ribosomal precursor RNA synthesis. Similar data were obtained when the RNA polymerases were assayed in isolated nuclei. These results indicate that the differential inhibition of RNA synthesis caused by 3'-dA in vivo cannot be simply explained by differential sensitivity of RNA polymerases I and II to 3'-dATP.     

Transcription of specific genes in isolated nuclei from HeLa cells in vitro.

Isolated HeLa cell nuclei were employed to catalyze the synthesis of RNA in vitro. In the presence of low concentrations of alpha-amanitin (1 mug/ml), used to suppress the formation heterogeneous nRNA, these nuclei synthesize RNA very efficiently for extended periods of time (at least 60 min) at an elongation rate of about seven nucleotides per second. The product, analyzed on sucrose density gradients and polyacrylamide gels was found to exist of two predominant size classes. Synthesis of the 45-S ribosomal precursor was completely resistant even to high concentrations of alpha-amanitin (150 mug/ml) and hence was catalyzed by enzyme A (or I). A limited degree of processing of the 45-S precursor occurred in vitro. In addition, a second RNA class of low molecular weight (4-8 S) was synthesized by HeLa cell nuclei in the presence of 1 mug/ml alpha-amanitin in vitro. Analysis on 8% polyacrylamide gels resolved the RNA into four distinct components. Their synthesis was resistant to low (1 mug/ml) but clearly sensitive to high (150 mug/ml) concentrations of alpha-amanitin. Consequently the synthesis of all these small-molecular-weight RNA species is catalyzed by RNA polymerase C (or III). For the assessment of the initiation frequency of the individual classes of RNA, a new technique was developed independent of labelling the 5' end of the RNA molecule with the gamma-phosphate of the initiating nucleotide. It employs the double labelling of an RNA molecule with two different isotopes added sequentially at different stages of completion of the chain. From the incorporation ratio of the two isotopes into a particular class of RNA, conclusions can be drawn concerning their initiation frequency. The results obtained have shown a high reinitiation frequency for the small-molecular-weight RNA species at all stages of the incubation reaction. In contrast, reinitiation of the 45-S precursor RNA occurs only to a limited extent in isolated HeLa cell nuclei in vitro.     

Isolation and partial characterization of the multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in the fungus Podospora anserina

Three DNA-dependent RNA polymerases have been isolated and partially purified from the mycelium of the fungus Podospora anserina. Separated by DEAE-Sephadex chromatography, they have been designated RNA polymerases I, II, and III according to their order of elution. Their catalytic properties and alpha-amanitin sensitivity are in agreement with those of the homologous enzymes found in other eukaryotic organisms. The three enzymes exhibit rather sharp monophasic ammonium sulfate dependence with optima which are, respectively, 0.035 M, 0.050 M, and 0.075 M. Enzyme I has the largest Mn2+/Mg2+ activity ratio, shows a marked preference for native DNA, and is insensitive to alpha-amanitin. Enzyme III uses poly(dA-dT) in preference to native DNA as template and is only partially sensitive to alpha-amanitin. Enzyme II is sensitive to alpha-amanitin, but high concentrations of the toxin are required for inhibition compared to other eukaryotic class II enzymes. Three similar RNA polymerases with comparable levels of activity were found in the temperature-dependent VR strain when cellular incompatibility, leading to a rapid cessation of RNA synthesis, was induced.     

Inhibition of yeast ribonucleic acid polymerases by thiolutin

Yeast ribonucleic acid (RNA) polymerase II, isolated after fractionation on diethylaminoethyl (DEAE)-cellulose (DE-52) or on DEAE-Sephadex (A-25), is 50% inhibited by 1.5 mug of alpha-amanitin. This inhibition is independent of the sequence of interaction of enzyme, template, nucleotides, and antibiotic and is expressed immediately on addition of alpha-amanitin to a preparation actively synthesizing RNA. Thus, alpha-amanitin's primary effect is inhibition of elongation of preinitiated RNA sequences in this system, as in others. A single peak of alpha-amanitin-resistant RNA polymerase activity (I) was eluted before enzyme II on either column. On A-25 but not on DE-52, a third peak of activity (III) was eluted after enzyme II. This activity was also resistant to alpha-amanitin. Enzymes I, II, and III were 50% inhibited by 3, 4, and 3 mug of thiolutin per ml, respectively. The extent of inhibition was independent of the nature of the template (native or denatured salmon sperm deoxyribonucleic acid or poly(dA-dT) or of the presence of 0.4 mM dithiothreitol, but this marked inhibition was only seen when enzymes were preincubated with thiolutin in the absence of template. Template protected the enzymes against thiolutin in the absence of nucleotides. Either the sensitive site on the polymerase is only accessible to thiolutin before interaction with template or thiolutin inhibits functional polymerase-template interaction but not elongation of preinitiated RNA chains.     

Three distinct forms of DNA-dependent RNA polymerase III from kidney

DNA-dependent RNA polymerases were extracted from nuclei isolated from 1 kg of pig kidney and subjected to DEAE-Sephadex chromatography using a step-wise salt gradient. Fractions corresponding to RNA polymerase III were pooled and rechromatographed on a second DEAE-Sephadex column using a linear salt gradient. At least three distinct peaks, designated as IIIA, IIIB, and IIIC were resolved. These peaks exhibited α-amanitin dose response curves characteristic of RNA polymerase III. Detection of the enzyme was facilitated by assaying with either highly polymerized calf thymus DNA and spermine or with poly [d(A-T)]. The heterogeneity of this enzyme became even more pronounced after further purification. Under the same conditions, both RNA polymerases I and II were resolved at most to two subspecies. The highly heterogeneous nature of RNA polymerase III is consistent with the large number of RNA species believed to be synthesized by this enzyme class.     

DNA-directed RNA polymerase from HeLa cells. Isolation, characterization and cell-cycle distribution of three enzymes.

DNA-directed RNA polymerase was solubilized from total HeLa cells. Three distinct classes of the enzyme could be clearly differentiated by their sensitivity toward alpha-amanitin. While form A is completely resistant to high concentrations (133 mug/ml) of this toxin, enzyme B is highly sensitive and is completely inhibited by concentrations of 0.1 mug/ml. In contrast, RNA polymerase C shows an intermediate behaviour (50% inhibition at 30% mug/ml). Separation of the three individual enzymes was achieved by chromatography on DEAE-cellulose (to separate enzyme B from A and C) and DEAE-Sephadex (to separate polymerase A from C). All three RNA polymerases were subsequently purified by phosphocellulose chromatography followed by sedimentation through glycerol gradients. Analysis of the purified enzymes by gel electrophoresis under denaturating conditions showed that the A enzyme consists of five subunits with molecular weights of 185, 128, 65, 41 and 32 X 10(3). In contrast, polymerase B is composed of seven subunits in variable stoichiometry with molecular weights of 215, 175, 145, 123, 68, 43 and 31 X 10(3) respectively. The subunit structure of enzyme C is not entirely clear at present and remains to be established. In addition, RNA polymerase activities were solubilized from mitotic and middle-S phase cells in comparison to controls. With respect to amounts and/or activities of all three RNA polymerases A,B and C no significant differences were detectable between logarithmically growing, mitotic and middle-S phase cells.     

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.     

DNA-dependent RNA polymerase from T8 Guerin tumor

DNA-dependent RNA polymerases from nuclei of T8 Guerin tumor were studied. Two enzymes were purified several hundred times by the use of ammonium sulfate precipitation, DEAE-cellulose and phosphocellulose chromatography. One of them belongs to A(I) RNA polymerases and the second to B(II) as was established from their metal and ionic strength requirements. activity in the presence of native and denatured DNA and the resistance to a-amanitin inhibition. The quantity of class A enzyme was increased compared to B, a fact observed with most neoplastic tissues so far studied. This increase of the polymerase responsible for ribosomal RNA synthesis could probably be related to malignant transformation in animals.     

Multiple forms of DNA-dependent RNA polymerases in Xenopus laevis. Rapid purification and structural and immunological properties

DNA-dependent RNA polymerases I, II, and III (EC 2.7.7.6) were isolated from Xenopus laevis ovaries. The soluble enzymes were precipitated with polyethyleneimine and subjected to chromatography on heparin-Sepharose, DEAE-Sephadex, and phosphocellulose. RNA polymerase I was subjected to an additional chromatographic step on CM-Sephadex. The procedure required 40 h and produced purified RNA polymerase forms IA, IIA, and III in yields of 5 to 40%. The specific activities of RNA polymerases IIA and III (on native DNA) were comparable to those reported from other eukaryotic sources, whereas that of form IA was severalfold greater than the specific activities reported for other purified class I RNA polymerases. The complex subunit compositions of chromatographically purified RNA polymerases IA, IIA, and III were distinct when analyzed by polyacrylamide gradient gel electrophoresis under denaturing conditions, although all three classes contained polypeptides with Mr = 29,000, 23,000, and 19,000. Antibodies prepared against RNA polymerase III showed common antigenic determinants within the class I, II, and III enzymes. The sites responsible for the cross-reaction are located, at least in part, on the common 29,000-dalton polypeptide.     

Synthesis of low molecular weight RNA components A, C and D by polymerase II in alpha-amanitin-resistant hamster cells.

In an attempt to establish which RNA polymerase catalyzes the synthesis of the low molecular weight RNA components A, C and D, Ama 1 cells (mutant Chinese hamster cells) were used in experiments with addition of alpha-amanitin. Ama 1 cells contain an altered RNA polymerase II which is 800 times more resistant towards inhibition by alpha-amanitin than the wild type enzyme. Alpha-amanitin (up to 200 microgram/ml) added to these cells does not affect the synthesis of the low molecular weight RNAs A, C and D. These data together with our previous data showing that alpha-amanitin (0.5 - 5.0 microgram/ml) preferentially inhibits the synthesis of A, C and D in normal cells indicate that RNA polymerase II catalyzes the synthesis of the low molecular weight RNA components A, C and D.     

Partial purification and characterization of DNA-dependent RNA polymerases I and II from cherry salmon (Oncorhynchus masou)

1. DNA-dependent RNA polymerases I and II were purified approx 3900- and 13,000-fold, respectively, from sonicated nuclear extract of cherry salmon (Oncorhynchus masou) liver by DEAE-Sephadex, heparin-Sepharose and DNA-cellulose column chromatography. 2. The purified RNA polymerases exhibited a requirement for four kinds of ribonucleoside 5'-triphosphates, an exogeneous template and divalent cation. 3. The activities of RNA polymerases I and II were inhibited by Actinomycin D (24 micrograms/ml) but not by Rifampicin (200 micrograms/ml). 4. RNA polymerase I preferred native DNA as template, while polymerase II preferred single-stranded DNA. 5. RNA polymerase II was inhibited by a low concentration of alpha-amanitin (0.02 micrograms/ml). RNA polymerase I was also inhibited by the relatively high concentration of alpha-amanitin (IC50 = 100 micrograms/ml and IC70 = 750 micrograms/ml). 6. RNA polymerases from cherry salmon exhibited a higher activity at low temperature than from rat liver.     

DNA-dependent RNA polymerase III from cauliflower. Characterization and template specificity

Class III DNA-dependent RNA polymerase (EC 2.7.7.6) was highly purified from cauliflower (Brassica oleracea, var. bortytis) by using polyethyleneimine precipitation. The specific activity of the enzyme was comparable to that reported for mammalian enzymes. Glycerol gradient sedimentation analysis indicated that the sedimantation coefficient (23 S) was slightly higher than that of enzyme II from cauliflower. The class III enzyme was inhibited by alpha-amanitin at high concentrations (50% inhibition at 200 microgram/ml). The Km value for nucleoside triphosphate was determined. Template specificities for single synthetic polymers showed that the enzyme read pyrimidine homopolymers as templates and preferred poly(dT) to poly(dC). The enzyme transcribed both strands of homopolymer pairs of poly(dI). poly(dC) and poly(dA).poly(dT). The synthetic polyribonucleotides were not effectively read. Competition experiments with these synthetic polymers indicated that the enzyme had different binding specificities which were not the same as their template specificities. The different binding affinities and template specificites for synthetic templates of the three classes of enzyme suggest that the enzyme can discriminate among different template sequences.