Showing papers on "RNA polymerase III published in 1974"
TL;DR: The role of RNA polymerase III (III(A) and/or III(B)) in the synthesis of (pre) 4S RNAs and a 5S RNA species is unequivocally demonstrated and rule out nonspecific inhibitory effects in the endogenous systems.
Abstract: Mouse myeloma cells have previously been shown (L. B. Schwartz, V. E. F. Sklar, J. A. Jaehning, R. Weinmann & R. G. Roeder, submitted for publication) to contain two chromatographically distinct forms of RNA polymerase III (designated IIIA and IIIB). The enzymes are unaffected by low α-amanitin concentrations which completely inhibit RNA polymerase II, but they exhibit characteristic inhibition curves (identical for IIIA and IIIB) at higher toxin concentrations. RNA polymerase I was unaffected at all α-amanitin concentrations tested. Myeloma RNA polymerases II, IIIA, and IIIB appear to be inhibited by the same mechanism, since the toxin rapidly blocks chain elongation by each enzyme. The characteristic α-amanitin sensitivity of RNA polymerase III has been employed in studies of the function(s) of the class III RNA polymerases.
Isolated myeloma nuclei and nucleoli continue to synthesize RNA via the endogenous RNA polymerases when incubated in vitro. With nuclei, newly synthesized 4S precursor (pre-4S) and 5S RNA species were detected by electrophoretic analysis either of the total nuclear RNA or of the RNA released into the supernatant during incubation. The synthesis of both pre-4S and 5S RNA species was inhibited by α-amanitin, but only at high concentrations; and the α-amanitin inhibition curves for these RNAs were identical to those obtained for solubilized RNA polymerases IIIA and IIIB. In control experiments it was shown that the endogenous RNA polymerase II activity of isolated nuclei was inhibited by α-amanitin concentrations similar to those required to inhibit purified enzyme II. However, 40-50% of the endogenous activity of nuclei and 100% of the endogenous activity of purified nucleoli was completely resistant to the high α-amanitin concentrations necessary to inhibit the RNA polymerase III activities. These experiments rule out nonspecific inhibitory effects in the endogenous systems.
These results unequivocally demonstrate the role of RNA polymerase III (IIIA and/or IIIB) in the synthesis of (pre) 4S RNAs and a 5S RNA species.
323 citations
TL;DR: The subunits α, β and β′, the constituents of core portion of RNA polymerase, were found to be produced coordinately during steady-state growth at different rates within the range examined and to be metabolically as stable as the bulk of protein.
Abstract: SummaryIn an effort to elucidate the control of synthesis of the DNA-dependent RNA polymerase in Escherichia coli, intracellular amounts of the individual subunits were determined by polyacrylamide gel electrophoresis of cell lysates and of precipitates formed with specific antibody against holoenzyme I.Polyacrylamide gel electrophoresis of cell lysates in the presence of sodium dodecyl sulfate has been believed to separate the two larger subunits, β and β′, of RNA polymerase from the bulk of protein in E. coli. However, a polypeptide unrelated to the polymerase was found to migrate in the immediate vicinity of the β′ subunit, interfering with the accurate measurement of this subunit. Taking account of the presence of this peptide, designated tentatively as χ, the quantity of RNA polymerase was estimated relying only on the β subunit.Subunits content was also measured by polyacrylamide gel electrophoresis of the precipitates formed by treating cell lysates with anti-holoenzyme I serum. Since the isolated individual subunits as well as the holo- and core enzyme could be precipitated by the antibody, the present procedure permitted to determine total amounts of the subunits within cells.The subunits α, β and β′, the constituents of core portion of RNA polymerase, were found to be produced coordinately during steady-state growth at different rates within the range examined (0.22 to 1.87 generations/hr) and to be metabolically as stable as the bulk of protein. The rate of synthesis of these subunits relative to the total protein was found to be balanced with the growth rate; the differential rate of synthesis of enzyme core (αp) can be represented by the following empirical equation: αp(%) = 0.7 μ + 0.45, where μ represents growth rate (generation/hr). In contrast, the content of σ subunit was considerably small, i.e. only about one third mole equivalent to enzyme core, and was almost unaffected by the rate of growth.
139 citations
TL;DR: A new form of DNA polymerase III, termed holoenzyme, has been purified to apparent homogeneity from gently lysed Escherichia coli and requires ATP to form an initiation complex with the primer template.
95 citations
TL;DR: The properties of the recently discovered mammalian cytoplasmic RNA polymerase activity is described with those of the animal nuclear enzymes because it is not yet known whether it is a true cytopLasmic enzyme, a nuclear enzyme that is leached out during homogenization, or a precursor of the nuclear enzymes.
Abstract: Publisher Summary In this chapter, the properties of the recently discovered mammalian cytoplasmic RNA polymerase activity is described with those of the animal nuclear enzymes because it is not yet known whether it is a true cytoplasmic enzyme, a nuclear enzyme that is leached out during homogenization, or a precursor of the nuclear enzymes. Purified B RNA polymerases (calf thymus, rat liver, or KB cells), calf thymus AI RNA polymerase, and rat liver AI and AII RNA polymerases sedimented through glycerol gradients at about 14-15 S faster than the E. coli core enzyme [MW 380,000-400,000]. B enzymes sediment slightly faster than A enzymes. These observations suggest a molecular weight of about 500,000. In contrast to E. coli RNA polymerase, there was no drastic modification of the sedimentation rate of animal enzymes as the ionic strength was varied. Molecular weights of 550,000 ± l0 %, 600,000 ± l0%, and 570,000 ± 10% were found for calf thymus AI, BI, and BII enzymes, respectively, by electrophoresis in polyacrylamide gels of increasing porosity. The molecular weight of the “cytoplasmic” RNA polymerase C from rat liver is probably similar because it was not resolved from the B enzymes by ultracentrifugation through sucrose gradients. Both AI and B enzymes are acidic protein, which migrates toward the anode at pH 8. After electrofocusing KB cell B enzymic activity peaked at around pH 4.74.
67 citations
TL;DR: Evidence is presented which strongly suggests that RNA polymerase C and the nuclearRNA polymerase III are one and the same enzyme.
50 citations
TL;DR: The tsX-mutation found in E. coli K12 strains Ts 19 and T 16 by a streptomycin method is a genetic marker for the @-subunit, and an enzymological comparison of the T16 enzyme with wildtype RNA polymerase reveals differences which might be due to the lesion in 0’.
44 citations
TL;DR: DNA-RNA hybridization-competition studies indicate that there are six temporal classes of RNA produced during the infection of Bacillus subtilis by bacteriophage SP82, and uninfected cell polymerase transcribes in vitro those DNA sequences which are expressed in vivo early in infection.
34 citations
TL;DR: Chromatin-bound RNA polymerase I and II from soybean hypocotyl can be separated by differential centrifugation and substitution of pH 6 for pH 8 isolation medium results in several-fold greater recovery of chromatin- bound RNA polymerases II.
29 citations
TL;DR: The results suggest that the polypeptide composition and the products of the cell-associated RNA polymerase are similar to those of the RNA transcriptase associated with influenza virus particles.
Abstract: The products synthesized in vitro by an RNA-dependent RNA polymerase isolated from influenza virus-infected BHK21-F cells were analyzed by velocity sedimentation, annealing techniques, and acrylamide-agarose gel electrophoresis. Approximately 50% of the RNA synthesized in vitro remains associated with the 50 to 70S ribonucleoprotein complex containing polymerase activity; the remainder of the RNA polymerase product sediments heterogeneously with a peak at 13S. At least 90% of the in vitro product hybridizes with virion RNA. If polypeptides are labeled early in the growth cycle, both the P and NP polypeptides are detected in the ribonucleoprotein complex by acrylamide gel electrophoresis. The results suggest that the polypeptide composition and the products of the cell-associated RNA polymerase are similar to those of the RNA transcriptase associated with influenza virus particles.
26 citations
TL;DR: The microsomal fraction, which most effectively converts aflatoxin B1 to an inhibitor of RNA polymerase is also the most effective cellular fraction catalysing the metabolism of aflat toxin B1.
Abstract: Aflatoxin B1 inhibits rat liver nucleoplasmic RNA polymerase B (40–50%) and cytoplasmic RNA polymerase C activities (25–35%) if applied in vivo. Nucleolar RNA polymerase A activity is not inhibited under the same conditions. Aflatoxin B1 has no effect on the activities of purified RNA polymerase enzymes A, B and C or on [3H]UTP incorporation of isolated rat liver nuclei in vitro. Aflatoxin B1, upon preincubation with a rat liver microsomal fraction, however, is apparently converted to a compound which then inhibits the activities of purified nucleoplasmic RNA polymerase (20–40%), cytoplasmic RNA polymerase (10–20%) and the incorporation of [3H]-UTP into isolated nuclei (38%). Nucleolar RNA polymerase activity is not affected under these conditions. The microsomal fraction, which most effectively converts aflatoxin B1 to an inhibitor of RNA polymerase is also the most effective cellular fraction catalysing the metabolism of aflatoxin B1.
25 citations
TL;DR: The results suggest that subunits of polymerase II obligatory to DNA-dependent RNA synthesis were degraded by “aging” and that a stable subunit of the RNA polymerase could preferentially make poly(A).
TL;DR: It is shown that the rifampicin-resistant mitochondrial RNA polymerase can be obtained in pure form from wild type and respiration deficient petite and raised the question as to whether these ‘complex mitochondrialRNA polymerases’ are in fact nuclear contaminants.
TL;DR: Starting nucleotide sequences of RNA synthesized using T7 DNA and E. coli RNA polymerase holoenzyme were determined and a ratio of the number of RNA started with ATP to that with GTP was 2.4 in the presence of 200 mM KCl.
Abstract: Starting nucleotide sequences of RNA synthesized using T7 DNA and E. coli RNA polymerase holoenzyme were determined. A ratio of the number of RNA started with ATP to that with GTP was 2.4 in the presence of 200 mM KCl, when more than 90% of the former had a unique sequence, pppApUpCpGp--and most of the latter had pppGpPyp--. On the other hand, at low KCl concentration, other sequences were observed.
TL;DR: The application of 5-formyluridine-5’-triphosphate (fo’ UTP) to the affinity labelling of RNA polymerase is reported, and the subunit involved has been reported.
TL;DR: Two RNA-dependent RNA polymerase activities are detected in the cytoplasm of influenza Ao/NWS infected cells; one activity has properties similar to the RNA- dependence polymerase activity associated with the influenza Ao-NWS virion and is stimulated by Mn2+ at high ionic strength.
Abstract: Two RNA-dependent RNA polymerase activities are detected in the cytoplasm of influenza Ao/NWS infected cells; one activity has properties similar to the RNA-dependent RNA polymerase activity associated with the influenza Ao/NWS virion and is stimulated by Mn2+ at high ionic strength. The other activity is stimulated by Mg2+ at low ionic strength. Both cellular activities are associated with a heavy complex which sediments heterogeneously in the region of monosomes in a sucrose gradient; this polymerase complex is not bound to ribosomes. The polymerase complex contains one main protein which corresponds to the nucleocapsid protein (NP) of the virus; it also contains segmented viral RNA. The polymerase complex and the ribonucleoprotein of the virus have similar components and sedimentation properties.
TL;DR: The effect of various concentrations of salt depends on the integrity of the viral DNA template suggesting some peculiarity of the transcriptional process of continuous double-stranded DNA by host RNA polymerases.
Abstract: I t has been shown tha t adenovirus 2 genes transcription involves host DNAdependent RNA polymerase I I (10, 13) which is compatible with the assumption tha t the adenovirus 2 virion does not introduce into the cell nucleus a DNAdependent RNA polymerase responsible for early gene transcription. In general, animal cell nuclei contain multiple DNA-dependent t~NA polymerases (6, 11, 14) originally designated ( l l ) as I, I I and I I I corresponding to forms A I /A II , 13 I / B I I and A I I I , respectively (7). RNA polymerase I is restricted to the nucleolus, whereas the RNA polymerases I I and I I I are in nueleoplasm (ll) , suggesting that the former synthesizes ribosomal RNA and the latter nuclear heterogeneous I~NA (16). Adenovirus 2 DNA is a defined template composed of a single piece of doublestranded DNA (2.3 • 107 daltons) which can be obtained in a homogeneous form from purified virions (2, 9). This communication describes the influence of ionic strength on the relative rate of adenovirus 2 DNA transcription in vitro by KB cell DNA-dependent t~NA polymerases I, I I and I I I . The ammonium sulfate concentrations required for optimal activity of KB cell RNA polymerases I and I I in the presence of intact adenovirus DNA are lower than those required for transcription of native KB cell DNA (1, 12). We show here tha t the effect of various concentrations of salt depends on the integrity of the viral DNA template suggesting some peculiarity of the transcriptional process of continuous double-stranded DNA by host RNA polymerases. The RNA polymerases were extracted from KB cell nuclei, as previously described (12). The enzymes were isolated by a DEAE-Sephadex A-25 column and elated with a linear gradient of 0.04=--0.5 N ammonium sulfate in a buffer contain-
TL;DR: On the basis of resistance to rifamycins and inhibition by α-aminitin, the soluble maizeRNA polymerase was shown to be equivalent to the nucleoplasmic RNA polymerase II of animals.
TL;DR: Two forms of DNA-dependent RNA polymerase were isolated from CV-1 cell cultures that are permissive to infection by SV40 and were partially purified by chromatographic fractionat.
Abstract: Two forms of DNA-dependent RNA polymerase were isolated from CV-1 cell cultures that are permissive to infection by SV40. RNA polymerases I and II were partially purified by chromatographic fractionat
Dissertation•
01 Jan 1974
TL;DR: In vitro incubation of oestradiol with uteri has shown that the stimulation of RNA polymerase activities in isolated nuclei is only slight when compared with the activities measured in nuclei obtained from uteri treated with oest radiol vivo, and both activities have been shown to be sensitive to the action of actinomycin D.
Abstract: 1. Several forms of DNA-dependent RNA polymerase have been purified from the uteri of immature rabbits. The isolation procedure involved the extraction of total uterine protein from a whole tissue homogenate using high salt concentrations. The RNA polymerases were partially purified by DEAE-cellulose chromatography and resolved into three species of enzyme which have been designated RNA polymerases A, B and C. These enzymes have been further purified by chromatography on phosphocellulose and by glycerol density gradient sedimentation. 2. The two major species of RNA polymerase, namely A and B, have been extensively characterised. Both enzymes sediment slightly faster than E. coli RNA polymerase in glycerol gradients suggesting a molecular weight in the range of 500,000 - 600,000. RNA polymerase A is more active in low concentrations of salt, although it can utilise both Mg++ and Mn++ efficiently, RNA polymers.se B is more active in high concentrations of salt with, Mn++ rather than Mg++ present as the divalent cation. RNA polymerase A is insensitive to the action of the toxin amanitin which specifically inhibits RNA polymerase B at similar concentrations. However, RNA polymerase A is more susceptible to thermal treatment than is RNA polymerase B. The template specificities of both enzymes have also been investigated. 3. A third species of enzyme, designated RNA polymerase 0, has been partially purified and characterised. This enzyme may be cytoplasmic in origin or may be 'soluble' with the result that it is leached out readily from the nuclei. RNA polymerase C has some properties similar to those of enzymes A and B and some which are intermediate between the two major enzyme species. 4. Two RNA polymerase activities have been identified in isolated nuclei; one has been equated with RNA polymerase A while the other has been equated with RNA polymerase Bo In vitro incubation of oestradiol with uteri has shown that the stimulation of RNA polymerase activities in isolated nuclei is only slight when compared with the activities measured in nuclei obtained from uteri treated with oestradiol vivo. 6. When measuring the endogenous RNA polymerase activities of isolated nuclei, prior treatment of the rabbits with oestradiol had a profound effect on the transcriptional capacity. Within 30-45 min after hormone treatment, the activity of RMA polymerase B was considerably increased. This activity decreased towards control levels at 1-2h before exhibiting a second increase of activity at about 3h. From 1h after oestradiol treatment, RNA polymerase A activity in the isolated nuclei was also increased and reached a plateau by about 4h. Both activities have been shown to be sensitive to the action of actinomycin D. 7. Treatment of the animals with amanitin prior to oestradiol inhibited the hormone-induced stimulation of RNA polymerase A as well as totally inhibiting RNA polymerase B. However, when amanitin was administered after the early enhancement of RNA polymerase B, the ocstradiol-induced stimulation of RNA polymerase A was retained. Treatment of the animals with cycloheximide prior to oestradiol did not affect the stimulation of RNA polymerase B but prevented the oestradiol-induced enhancement of RNA polymerase A. However, when cycloheximide treatment was delayed until after the early stimulation of RNA polymerase B, the activity of RNA polymerase A was stimulated. This suggested that stimulation of RNA polymerase A activity was dependent on protein synthesis subsequent to the hormone-induced stimulation of RNA polymerase B. 9. Since some indications were obtained of an effect of cytoplasm from oestradiol-treated rabbit uteri on the MA polymerase activity in nuclei, attempts were made to concentrate any such components. It was found that a fraction of the cytoplasm isolated from uteri treated with oestradiol for 30 rain was capable of stimulating RMA polymerase A activity in nuclei isolated from control animal uteri. 10. The isolated RNA polymerases from immature rabbit uteri do not show any increase in activity in response to oestradiol irrespective of the time of treatment with hormone. No fractions from cytoplasm treated with hormone have been shown to possess any stimulatory activity for either RNA polymerase A or B. It is possible that the observations of increased RNA polymerase activities in isolated nuclei result from changes in the transcriptional machinery rather than being due to alterations in the RNA polymerases per se.
01 Jan 1974
TL;DR: This talk will be the subject of this talk to review the work which led to these conclusions, and show that two separate DNA polymerases, called DNA polymerase (pol II) andDNA polymerase III (pol III), are similarly distinct from Pol I.
Abstract: When cells of an E. coli mutant (pol Al−)1 which is deficient in DNA polymerase I (pol I) are lysed in a French pressure cell, they yield a cell-free extract with a small but demonstrable amount of DNA polymerizing activity.2 Following the removal of nucleic acid from such an extract, phosphocellulose chromatography can resolve two separate DNA polymerases.3 Both of these DNA polymerases, called DNA polymerase (pol II) and DNA polymerase III (pol III), have been shown not only to be physically, catalytically, and genetically distinct from each other, but that they are similarly distinct from Pol I.4–6 It will be the subject of this talk to review the work which led to these conclusions.