Molecular Structures of DNA-Dependent RNA Polymerases (II) from Calf Thymus and Rat Liver

Weaver, Robert F.; Blatti, S. P.; Rutter, William J.

doi:10.1073/pnas.68.12.2994

Cited by 107 publications

(42 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Clearly, there is no evidence of any increase in the number of RNA polymerase molecules synthesising RNA in the presence of heparin as would be the case if blocked transcription complexes were being released. The value for the number of form B molecules measured in this study is in agreement with that published by Weaver et al [33] who estimated, from the specific activity of purified rat liver enzyme and the a-amanitin-sensitive activity in crude extracts, that 10000 molecules were present per nucleus. This value reduces to about 6500 molecules per diploid genome when corrected for the presence of tetraploid nuclei [22].…”

Section: Number Of Transcribing R N a Polymerase Moleculessupporting

confidence: 82%

The Mechanism by which Heparin Stimulates Transcription in Isolated Rat Liver Nuclei

Coupar

Chesterton

1977

European Journal of Biochemistry

View full text Add to dashboard Cite

The polyanion heparin, whilst inhibiting free RNA polymerase, stimulates the rate of RNA synthesis in eucaryotic nuclei ; it has been suggested that this process involves either the inactivation of RNase or the activation of blocked transcription complexes. These hypotheses have been investigated in isolated rat liver nuclei at 22 "C. We confirm that heparin inhibits free form A and B RNA polymerases but has little effect on the transcribing enzymes. The sensitivity of these enzymes to x-amanitin is unchanged by the agent and the stimulation is dependent on the presence of (NH4)2S04 and Mn2+ ions. The size of the RNA product was analysed on denaturing formamide/ sucrose gradients. It shows only a small increase when heparin is included in the incubation. In addition, no significant size changes are observed if the incubation is continued in the presence of actinomycin D and a-amanitin which together inhibit 97 % of all RNA synthesis. These results show that inactivation of RNase cannot be an explanation of the stimulatory effect in this system.Polynucleotide elongation rates and numbers of transcribing RNA polymerase molecules were determined by alkaline degradation of RNA labelled from [3H]UTP during 1 -min incubations. The [3H]UMP and [3H]uridine, derived from internal nucleotides and 3'-terminal nucleosides respectively, were separated by thin-layer chromatography and their tritium content measured. From this data the above parameters can be calculated. We show that the activity of RNase and phosphatase do not significantly affect the data and that levels of endogenous UTP are negligible. Data from eight separate experiments show that isolated rat liver nuclei contain 25-35 x lo3 transcribing RNA polymerase molecules per diploid genome comprising 18 -20 x lo3 form A and 5-15 x lo3 form B. Form C was not detected. Addition of heparin has no effect on these values. Polyribonucleotide elongation rates in nuclei are approximately 0.15 and 0.4 nucleotide per second for form A and B respectively. In the presence of heparin the latter rate is increased by 2-4-fold but that of the form A enzyme remains essentially unchanged. Similar results were obtained using [3H]GTP in place of [3H]UTP indicating that polyribonucleotide termination was random. Our findings show that heparin exerts its stimulatory effect by increasing the form-B-mediated polyribonucleotide elongation rate. It does not activate previously inactive RNA polymerase molecules.

show abstract

Section: Number Of Transcribing R N a Polymerase Moleculessupporting

confidence: 82%

The Mechanism by which Heparin Stimulates Transcription in Isolated Rat Liver Nuclei

Coupar

Chesterton

1977

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Comparison of the efficiency of our method with those reported by Stein and Hausen [9] and by Weaver et at. [17] for the solubilization and separation of the amanitinsensitive activity is impossible, since these authors did not state the yield of activity per weight of thymus. Applied to rat liver nuclei [15], our method was also more efficient in solubilizing the amanitinsensitive activity than those reported by other groups [18,19].…”

Section: Characteristics Of a And B Activitiesmentioning

confidence: 99%

Animal DNA‐Dependent RNA Polymerases

Kédinger

Gissinger

Gniazdowski

et al. 1972

European Journal of Biochemistry

109

View full text Add to dashboard Cite

This paper describes a large-scale method for the solubilization and the separation of calf thymus A and B DNA-dependent RNA polymerase activities. This method is reproducible, gives high yields and can handle up to several kg of tissue. The solubilization method involved homogenization of the whole tissue, sonication a t high ionic strength, ammonium sulphate precipitation and protamine sulphate precipitation to remove nucleic acids. The separation of the two types of activities A (insensitive to a-amanitin) and B (inhibited by this compound) was achieved by differential adsorption to DEAE-cellulose using a batch procedure. Studies of the inhibitory effect of a-amanitin on the various fraction during the solubilization and the separation stages suggest very strongly that the two types of activities pre-existed in vivo and did not result from the solubilization procedure.The separation of several peaks of DNA-dependent RNA polymerase activities on DEAE-cellulose or DEAE-Sephadex columns [1,2] and the differential effect of the inhibitor a-amanitin on these peaks [2] suggested that animal cells contained multiple RNA polymerases with specific intranuclear localizations [3]. Definite proof of the existence of several distinct enzymes in animal cells requires the purification of these activities and the demonstration that they correspond to different proteins. To carry out such studies, for which milligram quantities of highly purified enzyme are required, it was necessary to work out a large-scale purification procedure since the content of DNAdependent RNA polymerase in animal tissues, on a weight basis, is at the most 'Ilo of that of procaryotic cells (see Conclusion). This paper describes the first steps of such a procedure, which can be applied to kilogram quantities of tissue and which results in the separation of two types of activity; type A which is insensitive to a-amanitin and type B which is completely inhibited a t very low concentrations of this compound. Preliminary reports of this work have been published [2,4].

show abstract

“…Weaver et al (10) have suggested that RNA polymerase II of calf thymus is composed of four polypeptide chains of molecular weights 190,000, 150,000, 35,000, and 25,000. Kedinger et al (11,12) were clarified by high-speed centrifugation as described (5).…”

mentioning

confidence: 99%

RNA Polymerases of Maize. Purification and Molecular Structure of DNA-dependent RNA Polymerase II

Mullinix

Strain

Bogorad

1973

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Nuclear DNA-dependent RNA polymerase II has been purified from leaves of Zea mays by' a new procedure that improves enzyme stability and thus permits more manipulation during purification. The purification procedure includes a heating step, gel filtration on Sepharose 6B and 4B, and chromatography on DEAE-and DNAcelluloses. This method of purification yields an enzyme that exhibits maximal activity when denatured DNA is used as a template. Electrophoresis of highly purified enzyme on polyacrylamide gels containing sodium dodecyl sulfate indicates that maize RNA polymerase Ila is composed of several polypeptide subunits. The most highly purified preparations contain polypeptides with molecular weights of 200,000, 160,000, 35,000, 25,000,-20,000, and 17,000. were clarified by high-speed centrifugation as described (5). In a typical preparation, 4 kg of fresh weight of corn leaves yielded 5600 ml of clear amber supernatant (Fraction 1) after centrifugation of leaf homogenate at 100,000 X g for 90 min. The protein fraction that precipitated from this solution between 30 and 50% saturation of (NH4)2S04 was collected, dissolved in 150 ml of 50 mM Tris * HCl (pH 8.0)-20% glycerol-lOmM 2-mercaptoethanol (TGM) buffer and dialyzed against several changes of the same buffer. The solution was adjusted to a protein concentration of 25 mg/ml by addition of TGM (Fraction 2). It was heated to 50°for 10 min after which it was chilled and centrifuged at 50,000 X g for 20 min in a Type 30 rotor in a Spinco preparative ultracentrifuge; the supernatant was collected (Fraction 3).Sepharose Chromatography. Fraction 3 (130 ml) was applied to a 5.0 X 140-cm column of Sepharose 6B (Pharmacia Fine Chemicals,) equilibrated with TOM buffer. The column was washed with the same buffer at a flow rate of 90 ml/hr (Fig. 1). The tubes containing polymerase activity (e.g., fractions 51-70, Fig. 1) were pooled and concentrated by bringing the solution to 50% of saturation with (NH4)2S04. The pellet was dissolved in 30 ml of TGM buffer and dialyzed against the same buffer (Fraction 4). The solution was then applied to a 5 X 100-cm Sepharose 4B (Pharmacia Fine Chemicals) column from which the enzyme was eluted with TGM buffer at a flow rate of 25 ml/hr. The active fractions were pooled (e.g., fractions 75-95, Fig. 2) to give Fraction 5 (Table 1).DEAE-Cellulose Chromatography. Fraction 5 was applied to a DEAE-cellulose (Whatman DE-52) column prepared and equilibrated according to Burgess (13). RNA polymerase was eluted with a gradient of (NH4)2S04 in TGM buffer (Fig. 3). The active fractions were pooled (Fraction 6).Phase Partition Chromatography. Fraction 6 was further purified by phase partition chromatography according to Alberts (14). The phase containing RNA polymerase was exhaustively dialyzed against TGM buffer (Fraction 7).DNA-Cellulose Chromatography. Fraction 7 was applied to a DNA-cellulose column containing single-stranded calfthymus DNA prepared according to Alberts et al. (15) (Fig. 4)

show abstract

Molecular Structures of DNA-Dependent RNA Polymerases (II) from Calf Thymus and Rat Liver

Cited by 107 publications

References 14 publications

The Mechanism by which Heparin Stimulates Transcription in Isolated Rat Liver Nuclei

The Mechanism by which Heparin Stimulates Transcription in Isolated Rat Liver Nuclei

Animal DNA‐Dependent RNA Polymerases

RNA Polymerases of Maize. Purification and Molecular Structure of DNA-dependent RNA Polymerase II

Contact Info

Product

Resources

About