Eur. J . Biochem. 59,25-34 (1975)

Purification of Form A1 and A11 DNA-Dependent RNA Polymerases from Rat-Liver Nucleoli using Low-Ionic-Strength Extraction Conditions Barbara E. H. COUPAR and C. James CHESTERTON Department of Biochemistry, University of London King’s College, London (Received June 17, 1975)

Recent findings have confirmed the role of form A DNA-dependent RNA polymerase activity as that which is responsible for the transcription of the ribosomal RNA-coding genes. Unfortunately, the form A enzymes have proved to be very labile and difficult to work with, especially under high ionic strength conditions. We have, therefore, investigated a method for the purification of the form A1 and A11 enzymes from rat liver using mild low-ionic-strength conditions. Since preparations from whole nuclei were found to be grossly contaminated with protein having similar properties, the enzymes are extracted from nucleoli. Forms A1 and A11 are separated on a phosphocellulose column, purified by further ion-exchange chromatography, and by sedimentation through a glycerol gradient. The purified enzymes each migrate as a single band on native polyacrylamide gels and have the expected characteristics of form A RNA polymerase. Sedimentation rates through glycerol gradients indicate that they both have a similar size to that of Escherichia cofi RNA polymerase ( M , about 500000). The purified enzymes are free of DNase and RNase. A method is also described for the purification of form B from the nucleoplasm remaining after isolation of nucleoli. The presence of form C activity was not detected.

It can now be stated that a characteristic property of eukaryotes is the presence of form A (or I), form B (or 11), and form C (or 111) DNA-dependent RNA polymerases [l -41. They are easily distinguished by their relative sensitivities to the fungal toxin a-amanitin [ 5 ] , whereas form A activity is totally insensitive, form B is inactivated at a low concentration of the drug (1 pg/ml) and form C has intermediate properties (200 pg/ml is required for inhibition). Form A activity has been shown to be located in the nucleolus, whereas forms B and C are found in the nucleoplasm. From these locations and other evidence [6- 111 it has been assumed that form A reads ribosomal RNA-coding genes and form B the mRNA-coding template. Form C activity has recently been shown to be responsible for the synthesis of 5-S RNA and tRNA precursor-RNA [2]. Beebee and Butterworth [12- 141 have confirmed the role of form A RNA polymerase, using enzyme and template from Xenopus luevis ovaries. Support for the role of form B was demonstrated by ButterEnzyme. RNA polymerase or nucleoside triphosphate RNA nucleotidyl transferase (EC 2.7.7.6).

worth et a f . [15] when it was shown that carefully prepared chromatin is read only by this type of RNA polymerase. The importance of these enzymes in the selective control of gene expression is therefore clear. Despite intensive research, the mechanism controlling ribosomal RNA synthesis in prokaryotes [16] and eukaryotes is still unclear [lo, 111. We reported previously [17,18] the separation of two form A DNAdependent RNA polymerase activities called A1 (or I a) and A11 (or I b). These enzymes are obviously of great interest in relation to the transcriptional control of ribosomal RNA synthesis. We have, therefore, developed a procedure for their further purification. Our previous method involved extracting the enzymes from intact nuclei. However, owing to the well known instability of the form A enzymes and gross contamination with other similar nuclear proteins, in spite of repeated efforts, the purification could not be improved beyond the point reported. The only extensive purification of form A reported in the literature is that of Gissinger and Chambon [19] who extracted the activity from calf thymus. Form A11

26

was not detected. We report here the use of rat liver nucleoli as an enriched source of the enzymes. A procedure has been developed for the purification of form A1 and A11 to near homogeneity. A method for extracting form B from the nucleoplasm which remains after separation of nucleoli is also described. We have not detected form C in rat liver nuclei prepared, as here, by sedimentation through 2.4 M sucrose.

MATERIALS AND METHODS Biochemicals

Materials were obtained from the following companies : [3H]UTP from the Radiochemical Centre (Amersham, England), UTP, CTP and GTP (trisodium salts) from Boehringer Mannheim (Mannheim, Germany); ATP, DNase I, pancreatic RNase, actinomycin D, Coomassie brilliant blue and phenol red from Sigma Chemical Co. ; dithiothreitol from KochLight ; butyl-PBD from Intertechnique; Analar glycerol, acrylamide and bis-acrylamide from British Drug Houses. DEAE-cellulose (DE-23) and phosphocellulose (P11) were obtained from Whatman and washed as previously described [17]. a-Amanitin and rifamycin AF/O13 were the generous gifts of Professor T. Wieland and Professor L. G. Silvestri respectively. Standard Assay for RNA Polymerase

Enzyme activity was determined by measuring the amount of [3H]UTP converted into acid-precipitable RNA essentially as described previously [17]. Incubations contained 25 mM Tris-HC1 pH 8, 200 pg/ml bovine serum albumin, 1 mM dithiothreitol, 0.3 mM each ATP, CTP and GTP, 0.04 mM [3H]UTP 49 pCi/ pmol, 40 pg/ml rat liver DNA [20] and enzyme samples of 0.05 ml, 0.02 ml or 0.01 ml in a total volume of 0.25 ml. Assays for form A RNA polymerase activity contained in addition 6 mM MgC12 and either 0.18 M KCl or 0.04 M (NH4)2S04[17,18]. Form B assays contained 1 mM MnC12 and 0.13 M (NH4)&304 [18,21]. After incubation, normally for 10 min at 37 "C, the assay mixture was cooled to 0 "C and 1 ml 10 % trichloroacetic acid containing 5 % (w/v) N%P207 added. After 10 min at O"C, the acid-precipitable material was collected by filtration on a Whatman GF/C 25-mm-diameter glass fibre filter and washed successively with 50 ml ice-cold 5 % trichloroacetic acid containing 3 % Na4P20, and a small volume of water to remove the acid. The filters were dried under an infrared lamp and put into vials with 3 ml toluene containing 6 g butyl-PBD per litre. The radioactivity present was determined at a counting efficiency of 24.6% in a Tracerlab Corumatic 200 scintillation spectrophotometer. One enzyme unit was defined as

Eukaryotic RNA Polymerases

the incorporation of 1 pmol of UMP per min incubation, and enzyme specific activity was expressed in units/mg protein. Protein and DNA were determined as described previously [151. Purification of Form A RNA Polymerases Preparation of Nuclei. All procedures were carried out at 4°C unless stated otherwise. Nuclei were prepared from approximately 600 g rat liver as described by Chesterton and Butterworth [17], with the omission of KCl from both the sucrose homogenising medium and the 2.4 M sucrose solution. The enzyme purification procedure is outlined in Scheme 1. Preparation of Nucleoli and Nucleoplasm. Nucleoli were prepared by the Busch method [22]. After sedimentation through 2.4 M sucrose [17], nuclei were resuspended in 0.25 ml of 0.32 M sucrose containing 0.5 mM dithiothreitol (sonication medium) per g original liver. This was achieved by homogenisation as described previously [ 171. The resuspended nuclei were sonicated in 20 ml batches for 4 x 10 s, with 20 s cooling between each burst, at medium power with the large probe of the MSE sonicator or until the number of nuclei in any one microscope field at 400-fold magnification was less than 4-5. The glass vessel holding the nuclear suspension was cooled in ice water. The sonicate was then layered over an equal volume of 0.88 M sucrose containing 0.5 rnM dithiothreitol and spun at 2000 x g (3250 rev./min) for 30 min in the MSE Mistral 6L. The nucleoplasmcontaining supernatant was adjusted to 1 M sucrose, 0.01 M Tris-HC1 pH 8,O.l mM EDTA, 5 mM MgC12 and 1 mM dithiothreitol. This was stored at - 70°C until required for the preparation of form B RNA polymerases as described below. Buffers. Extraction buffer contained 25 % glycerol (v/v), 0.01 M Tris-HC1 pH 8, 0.1 mM EDTA, 5 mM MgC1, and 0.5 mM dithiothreitol together with KCI at the stated concentration. Glycerol buffer A contained 0.01 M Tris-HC1 pH 8.0, 0.1 mM EDTA and 0.5 M dithiothreitol together with glycerol at the stated concentration. DEAE-Sephadex buffer contained 0.05 M Tris-HC1 pH 8.0, 0.1 mM EDTA, 40% glycerol and 0.5 mM dithiothreitol. Glycerol buffer B contained 0.05 M Tris-HC1 pH 8.0, 5 mM MgCl,, 0.1 mM EDTA and 1 mM dithiothreitol together with the stated concentration of glycerol. Glycerol phosphocellulose buffer B contained 0.05 M Tris-HC1 pH 8, 1 mM MgC12, 0.1 mM EDTA and 1 mM dithreitol together with the stated concentration of glycerol. Low Salt Extraction of Form A RNA Polymerases from Nucleoli. The nucleoli pellets were resuspended with 10 ml 0.07 M KCl extraction buffer and incubated at 37 "C for 30 rnin with shaking to maintain

21

B. E. H. Coupar and C . J. Chesterton

an even suspension. The nucleoli were spun down at 1800 x g (2400 rev./min) for 10 min in the MSE Mistral and the supernatant (0.07 M KCl extract) was adjusted to 40 % glycerol. A further extraction was carried out similarly at 0.12 M KC1 using 5 mlO.12 M KC1 extraction buffer, and the nucleoli were spun down at 12000 x g (loo00 rev./min) for 15 min in the MSE 18. The supernatant (0.12 M KCl extract) was also adjusted to 40% glycerol. Both extracts were stored at - 70 "C until required, as were all samples and fractions at subsequent stages of purification. DEAE-Cellulose Chromatography.The 0.07 M KC1 and 0.12 M KCI extracts were combined and passed at 80 ml/h through a 12 x 1.5-cm column of DEAEcellulose, previously equilibrated with 0.05 M KC1 25 % glycerol buffer A. After loading, the column was washed successively with 0.05 M KCI 40% glycerol buffer A and 0.1 M KC1 40% glycerol buffer A. In each case the level of protein, as monitored by an LKB Uvicord I1 at 279 nm, was allowed to return to near the background level. The enzyme was eluted with 0.23 M KC1 40% glycerol buffer A and 1.8-ml fractions were collected. A further wash of 0.4 M KCl 40 % glycerol buffer A was applied. The fractions were assayed for RNA polymerase (0.02-ml samples) and those containing the peak of activity were combined. This fraction was termed the form A DEAEcellulose enzyme (see Scheme 1). Phosphocellulose Chromatography. DEAE-cellulose enzyme (usually about 10 ml) was loaded on to a 6 x 1.5-cm column of phosphocellulose, equilibrated with 0.05 M KC1 40 % glycerol buffer A, at 40 ml/h. The column was washed with 0.2 M KCI 40% glycerol buffer A until no more protein was eluted and a 60-ml linear gradient from 0.2 M KC1 to 0.8 M KC1 40% glycerol buffer A was applied. This was immediately followed by a 15-ml wash with 0.8 M KCl 40% glycerol buffer A. In later preparations a 60-ml linear gradient from 0.2 M KCl to 1 M KCl 40% glycerol buffer A was used with no additional wash. This achieves the same result. 1.8-ml fractions were collected, 100 pg bovine serum albumin was added to each unless specified otherwise. Aliquots of 0.02 ml were assayed for RNA polymerase activity. Fractions from the two peaks of activity were combined as phosphocellulose-1 enzymes A1 and A11 respectively. The A11 phosphocellulose-1 enzyme was applied to a second phosphocellulose column exactly as before after 2 h dialysis at 0 ° C against 2 1 0.05 M KCI, 0.01 M Tris-HC1 pH 8, 0.1 mM EDTA, 25 % glycerol and 15 mM 2-mercaptoethanol and dilution with an equal volume of 40 % glycerol buffer A containing no KCl. Fractions were assayed as before and the peak of activity (A11 phosphocellulose-2 enzyme) was combined. DEAE-Sephadex Chromatography. After dialysis for 3 h at 0°C against 0.05 M KCI, 0.05 M Tris-HC1

pH 8, 0.1 mM EDTA, 25% glycerol and 15 mM 2-mercaptoethanol, the phosphocellulose enzyme was loaded on to a 10 x 1.0-cm column of DEAE-Sephadex A-25, previously equilibrated with 0.05 M KCI 25 % glycerol buffer A, at 80 ml/h. The column was washed with 0.15 M KC1 DEAE-Sephadex buffer and then with 0.35 M KCI DEAE-Sephadex buffer to elute the enzyme. Fractions of 0.6 ml were collected and assayed for RNA polymerase activity (0.01-ml samples). The fractions from the peak of activity were combined and referred to as A1 or A11 DEAESephadex enzyme. Ammonium Sulphate Precipitation. DEAE-Sephadex enzymes were dialysed for at least 6 h or overnight at 0 "C against saturated ammonium sulphate pH 8, 0.05 M Tris-HC1 pH 8, 0.1 mM EDTA and 15 mM 2-mercaptoethanol. The dialysate was then spun at 110000 x g (40000 rev./min) for 15 min in the 10 x 10 titanium rotor of the MSE 65 ultracentrifuge. Supernatants were decanted. The centrifuge tubes were drained over tissue and wiped dry. The precipitates (A1 and A11 ammonium sulphate enzyme) were stored at - 70°C until required for loading on to glycerol gradients. Glycerol Gradient Centrifugation. 6-ml linear gradients of 15% to 30% glycerol containing 0.05 M Tris-HCI pH 8, 0.1 mM EDTA, 0.1 M (NH4)2S04r 5 mM MgC1, and 1 mM dithiothreitol were prepared and allowed to equilibrate at 4°C overnight before use. Each (NH,),SO,-precipitated pellet was dissolved in approximately 0.1 ml of 0.05 M Tris-HCl pH 8, 0.1 mM EDTA, 0.1 M (NH4)2S04, 5 mM MgCl,, 1 mM dithiothreitol and layered on top of a gradient. Each was spun at 250000 x g (60000 rev./min) for 7 h at 4°C in the 3 x 6.5-ml titanium rotor of the MSE 65 centrifuge. Fractions of 0.3 mi were collected from the bottom of the tube and assayed for RNA polymerase activity (0.01-ml samples). The peak of activity was referred to as A1 or A11 glycerol-gradient enzyme. Protein was determined by measurement of absorbance at 280 nm. The linearity of the gradients was checked by refractive index measurements. Purification of Form B RNA Polymerase from Nucleoplasm Extraction of Form B Activity. Nucleoplasm in 80-ml batches was adjusted to 0.35 M (NH4),S04 by the addition of 4 M (NH4),S04 and sonicated at full power for 10 x 10 s (waiting 20 s for cooling between bursts) with the medium probe of the MSE sonicator. It is important that the glass vessel holding the nucleoplasm be cooled in ice water. The sonicate was diluted with 2 vol. of 25 % glycerol buffer B and spun at 23000 x g (11 000 rev./min) for 20 min in the MSE 18. The pellet was discarded and 0.42 g (NH4)2S04 per ml added slowly to the supernatant

28

Eukaryotic RNA Polymerases

Rat liver nuclei sonication centrifugation

1

7 Nucleoplasm

Nucleoli

I

+

I

0.07 M KC1, 37" C

Extracted nucleoli

sonication (NH+S04 pptn dialysis

4

0.12 M KCl, 37" C

Form B enzyme nucleoli

0.07M KCl

extract

extract

DEAE-Sephadex

4

1

DEAE-cellulose

B DS enzyme

I'

Form A DC enzyme

phosphocellulose

phosphocellulose

I

f

A11 PCl, enzyme

1

B PC enzyme

I

dialysis PC2

1

A

A1 PCl enzyme

A1 PC2 enzyme

1

dialysis DEAE-Sephadex A1 DS enzyme

I I

A11 PC2 enzyme

+

B DC enzyme

dialysis DEAE-Sephadex A11 DS enzyme

I I

(NH+S04 pptn

(NHdzS04 pptn

A1 AS enzyme

A11 AS enzyme

glycerol gradient

glycerol gradient

A1 GG enzyme

DEAEcellulose

A11 GG enzyme

Scheme 1. Procedure,for rhepurificarion oflorm A l , A l l and B R N A polyrnerasesfiom rur liver nuclei. DC cellulose; DS = DEAE-Sephadex; GG = glycerol gradient; AS = (NH,),SO,

at 0°C. When the ammonium sulphate was dissolved, the solution was spun as before and the supernatant discarded. The ammonium-sulphate-precipitated pellet was resuspended in a minimal volume of 0.04 M (NH4)2S0425 glycerol buffer B and dialysed overnight against 8 1 of 0.04 M (NH4)2S0425 % glycerol buffer B at 0 "C. The dialysate was spun at 38 000 x g

=

DEAE-cellulose; PC

=

phospho-

(18000 rev./min) for 20 min and pelleted material was discarded. DEAE-Sephadex Chromatography. Dialysate derived from approximately 300 g liver was diluted with an equal volume of glycerol buffer B before loading at 80 ml/h on to a 35 x 2.5-cm column of DEAESephadex A-25, equilibrated with 0.04 M (NH4)2S04

29

B. E. H. Coupar, and C. J. Chesterton

25 "/, glycerol buffer B. After loading, the column was washed with 50 ml 0.04 M (NH4)2S0425% glycerol buffer B and a linear gradient from 0.04 M (NH4),S04 to 0.5 M (NH4);S04 in 180 ml 25 % glycerol buffer B was applied, followed by a further 50-ml wash of 0.5 M (NH4),S04 25 "/, glycerol buffer B. Fractions of 3 ml were collected and assayed for RNA polymerase activity (0.05-ml samples). The active fractions were combined to yield the form B DEAESephadex enzyme (see Scheme 1). Phosphocellulose Chromatography. Form B DEAESephadex enzyme was dialysed for 3 h at 0 "C against 2 1 of 25 yo glycerol phosphocellulose buffer B and then diluted with an equal volume of 25% glycerol phosphocellulose buffer B. This solution was loaded at 40 ml/h on to a 6 x 1.5-cm column of phosphocellulose, previously equilibrated with 0.05 M (NH4)2S0, 25 % glycerol phosphocellulose buffer B. The column was washed with 0.05 M (NH4&304 40 glycerol phosphocellulose buffer B until the absorbance at 279 nm returned to a background level. A 50-ml linear gradient from 0.05 M (NH4),S0, to 0.5 M (NH4),S04 40 7; glycerol phosphocellulose buffer B was applied and 1.2-ml fractions were collected. The fractions were assayed for RNA polymerase activity (0.02-ml samples) and active fractions were combined (form B phosphocellulose enzyme). DEAE-cellulose Chromatography. Form B phosphocellulose enzyme was dialysed for 2 h against 0.05 M (NH4)$04 25 2,glycerol phosphocellulose buffer B at 0 "C and diluted with 0.5 vol. 40 % glycerol phosphocellulose buffer B. The dialysed, diluted enzyme was applied at 40 ml/h to a 5 x 1.0-cm column of DEAE-cellulose. previously equilibrated with 0.05 M (NH4),S04 40 % glycerol phosphocellulose buffer B. The column was washed with 0.1 M (NH4)2S04 40 % glycerol phosphocellulose buffer B until no further protein was eluted. The enzyme was then eluted with 0.35 M (NH4)2S04 40% glycerol phosphocellulose buffer B. Fractions of 0.6 ml were collected and assayed for RNA polymerase activity (form B DEAE-cellulose enzyme) as above.

x,

Po!,-acrylamide Disc Gel Electrophoresis

Polyacrylamide gels (4%, 6 x 0.5 cm) were made up as follows: 20 ml 0.2 M phosphate buffer pH 7.4, 4.8 ml 33.3",, acrylamide, 3.2 ml 1.30/(,his-acrylamide and 11.7 ml water were mixed, deaerated and 0.3 ml 10% ammonium persulphate and 0.02 ml N,N, N',N'tetramethylethylenediamine were added. The mixture was dispensed into glass tubes (7 x 0.5 cm bore), covered with a layer of water and allowed to set for at least 2 h. They were normally stored in the dark at room temperature until required. Gels were prerun for 2 h with 0.1 M phosphate buffer, pH 7.4, 15 mM 2-mercaptoethanol at 5 mA per gel and 4 "C before

samples were loaded. Samples and standards were adjusted to equal volumes and mixed with 5 p1 1 M dithiothreitol and 5 pl 0.2% phenol red. All contained at least 25 % (v/v) glycerol. Gels were then subjected to electrophoresis as before until the phenol red was approximately 1 cm from the bottom. On removal the dye front in the gel was marked with wire. Gels were stained overnight at room temperature with 0.25 % Comassie brilliant blue, 2.5 glycerol, 40% methanol and 5 % acetic acid and subsequently destained with 7.5 % acetic acid and 10 methanol at 37 "C. TestsJor Nuclease in the Enzyme Preparations Ribonuclease. Enzyme samples were tested for the presence of ribonuclease under standard RNA polymerase assay conditions by comparing the acidprecipitable radioactivity remaining after tritiumlabelled yeast RNA (1.1 pg) was incubated for 10 rnin at 37°C in the presence or absence of enzyme. The test is capable of detecting the presence of 0.01 pg pancreatic RNase. Deoxyribonuclease. HeLa [3H]DNA (0.7 pg) was incubated for 10 rnin at 37°C in the presence of enzyme under normal RNA polymerase assay conditions. An equal volume of 0.6 N NaOH was added and the incubation continued for a further 10 min at 37" C. The incubation mixtures were then loaded on to 6-ml gradients of 10-30% sucrose containing 0.1 M NaOH, 0.9 M KCl and 0.1 M EDTA and spun at 250000 x g (60000 rev./min) for 3 h at 4 "C in the 3 x 6.5-ml titanium rotor of the MSE 65 ultracentrifuge. Fractions of 0.3 ml were collected from the bottom of the tube, precipitated with 1 ml 10% trichloroacetic acid and counted for radioactivity. The distribution of radioactivity in the gradient was compared with that obtained for a similar incubation containing no enzyme.

RESULTS AND DISCUSSION Extraction of'Form A R N A Polymeruses from Nucleoli

Preliminary tests indicated that maximal yields of DNA-dependent activity could be obtained by leaching out the enzymes at 37 "C. However, this extraction proved to be a complex process. Fig.1 shows the release of form A activity with time of 37'C incubation at various KCI concentrations. All enzyme assays were performed at 0.18 M KCI. Maximal activity is extracted, using 0.05 M KCI for 60 rnin or 0.1 M KCl for 30 min. However, it was found that increased yields resulted by using a sequential cxtraction: 30 rnin at 0.05 M KCI plus 30 rnin at 0.1 M KCI. A further 30 rnin in 0.15 M KC1 did not improve the yield significantly. The result suggested a bimodal

30

Eukaryotic RNA Polymerases

-s 100 u 2 80 0 L L

X

e2

60

8

40

L

0.05

0.10

0.15

W I l (MI

I . . . . . . . 0 I0 20 30 50 60 LO

lime I min)

Fig. 1. Extraction of R N A polymerase activityfrom nucleoli at various times and salt concentrations. Batches of nucleoli each derived from 47 g of rat liver were resuspended with 0.4 ml of buffer containing 0.05M Tris-HCI pH 8, 0.1 mM EDTA, 5 m M MgCl2. 25% glycerol, 1 mM dithiothreitol and either 0.05 M KCI (O), 0.1 M KCI (0)or 0.15 M KCI (A) and incubated at 37°C. 0.1-ml samples were withdrawn after 0, 10, 30 and 60 rnin and spun at 1800 x g (2400 rev./min) for 20 min in the MSE mistral. Duplicate 10-pl portions of the supernatant were assayed for RNA polymerase activity as described in Materials and Methods. Sequential extractions were performed at 0.1 M KCI (0)and 0.15 M KCI (W) on the pellets from equivalent batches of nucleoli which had been extracted at 0.05 M KCI for 30 rnin and 0.05 M KC1 for 30 rnin plus 0.1 M KCI for 30 min respectively. Values for total polymerase activity extracted are shown

salt effect, which was confirmed by the results shown in Fig.2. The effect of KCl concentration on the extraction of form A from nucleoli was tested. The activity recovered was highest at 0.07 M KCl, lowest at 0.09 M KCl and rose to a second peak at 0.12 M KCI. If the nucleoli extracted at 0.07 M KCl were re-extracted at 0.12 M KCl, the second peak of activity could be recovered. This final procedure adopted, therefore, involved a sequential extraction at 0.07 M KCI and 0.12 M KC1 as described in Methods. The explanation of this phenomenon which apparently relates to the differential properties of forms A1 and A11 will be discussed in a subsequent paper. Separation and Purification of Forms AI and AII by Ion-Exchange Chromatography

Fig.3 shows the stepwise elution of the form A activity from a DEAE-cellulose column. As reported

Fig.2. Effect of KCI concentration on the extraction of RNA polymerase acfivityfrom nucleoli. Each aliquot of nucleoli was derived from 6 g of rat liver and was extracted with 0.4 ml of buffer containing 0.05 M Tris-HCI pH 8, 0.1 m M EDTA, 5 mM MgCI,, 25 % glycerol and 0.5 mM dithiothreitol at KC1 concentrations from 0.05 M to 0.15 M KCI (0).Nucleoli were incubated at 37 "C for 30 min, spun at 320 x g (lo00 rev./min) for 5 rnin in the MSE mistral. Samples of nucleoli before extraction and the supernatants (extracts) were assayed for RNA polymerase activity. The pellets from the 0.05 M, 0.06 M and 0.07 M KCI points were combined and re-extracted for 30min at 37°C with the same buffer at KC1 concentrations from 0.08 M to 0.15 M (0)

for the nuclear-extracted enzymes [17], the RNA polymerase activity is eluted between 0.1 M and 0.23 M KCl. When this fraction is loaded on to a phosphocellulose column and eluted with a linear gradient of KCl, two peaks of RNA polymerase activity, termed A1 and AII, are eluted at approximately 0.5 M and 0.65 M KCI respectively (Fig.4A). Due to the low protein concentration after this step, the enzymes were stabilized by addition of bovine serum albumin. When the A1 and A11 peaks were rerun under similar conditions, their position of elution was unchanged (Fig. 4 B). Form A11 was routinely rerun on phosphocellulose to remove contaminating A1 activity. The two enzymes were further purified and concentrated by stepwise elution from DEAE-Sephadex as shown in Fig. 5. Sedimentation of Forms AI and A l l through a Glycerol Gradient

Both forms A1 and A11 sediment at a rate similar to that of purified Escherichia coli RNA polymerase (Fig.6), which is known to have a molecular weight 0f480000-500000 [23]. Purity and Basic Properties of the Enzymes

Tables 1 and 2 show the degrees of purification attained, for A and B RNA polymerase respectively, at each step of the procedures described above. The

31

B. E. H. Coupar and C. J. Chesterton

5 1015202530 Fraction number Fig. 3. Chromatography of form A RNA polymerase on DEAE-cellulose. Combined 0.07 M KCI and 0.12 M KCl extracts of nucleoli containing 3800 enzyme units of RNA polymerase activity were subjected to chromatography on DEAE-cellulose. Enzyme units of RNA polymerase activity in the 0.23 M KCI eluate fractions are shown (0).The eluates from the 0.05 M KCI, 0.1 M KCI and 0.15 M KCI washes contained 60,70 and 600 enzyme units of RNA polymerase activity respectively. 6300 enzyme units of RNA polymerase activity at 280 nm (protein); (----) KCl concentration were recovered from the combined peak fractions of the 0.23 M KCI eluate. (-)Absorbance 0

‘I..

150(

A

1

’50 1.0

I

1-1__,_.’------. 0.8

.

loo

1: 0.5

E

Enzyme

~~

r

0.6

I

Y

~. -. ...

0.8

5 10 15 20 25 30 35 LO Fraction number Fig.4. Chromatography of form A RNA polyrnerases on phosphocellulose. (A) Form A DEAE-cellulose enzyme (9400 units) was

0

subjected to chromatography on phosphocellulose. RNA polymerase activity in the flow through and 0.2 M KCI wash was 40 enzyme units, and 450 and 2700 enzyme units of form A1 and form A11 RNA polymerase activity respectively were recovered in ) polymerase activity. the gradient fractions. (MRNA (9) Form All phosphocellulose-1 enzyme (2700 units) was rechromatographed on phosphocellulose after dialysis and 1040 enzyme units(-) were recovered. When 960 units of form A1 phosphocellulose-1 enzyme were rechromatographed o n a similar phosphocellulose column, the distribution of RNA polymerase activity was as shown (M and ) 620 units were recovered from the combined peaks fractions. (-) Absorbance at 280nm, (------)KCI concentration

Fig. 5 . Chromatography of form A1 and form A l l RNA poljmiwses on DEAE-Sephadex. (A) Form A1 phosphocellulose enzyme (640 units) was subjected to chromatography on DEAE-Sephadex. The flow through and 0.15 M KCI wash contained a total of 20 enzyme units of RNA polymerase activity. and 580 units were recovered from the combined peak fractions of the 0.35 M KC1 eluate. (B) Form A11 phosphocellulose enzyme (2200 units) was subjected to chromatography on DEAE-Sephadex. The flow through and 0.15 M KC1 eluate contained 30 enzyme units of RNA polymerase activity, and 1500 enzyme units were recovered from the combined peak fractions of the 0.35 M eluate. ( 0 4 ) RNA polymerase absorbance at 280 nm; (------)KCI concentration activity; (--)

Eukaryotic R N A Polymerases

375 X W

73

370 C

-8

W .-

365 t, [L

1

5

10 15 Fraction number

20

Fig.6. Glycerol gradient cmrrifixution o j j o r m A1 and form AII R N A polymerases. Form A1 (400 units) and form A11 (700 units) RNA polymerases concentrated by ammonium sulphate precipitation were subjected to glycerol gradient centrifugation as described in Methods. Fractions (0.3 ml), numbering from the bottom of the gradient, and the pelleted material resuspended with 0.3 ml of the 30:',, glycerol gradient buffer were assayed for R N A polymerasc activity. The distribution of form A1 ( 0 - 4 )and form A11 (t-) RNA polymerase activity is shown. The recovery of RNA polymerase activity from the gradient was 150 enzyme units for form A1 and 200 enzyme units for form AII. Similar gradients were used for refractive index measurements ( - - - - - - ) and to determine the position of sedimentation of E. coli RNA polymerase (indicated by the arrow) under thc same conditions. A typical distribution of protein through the gradient is shown (--)

Fraction number

Fig. 7. Ion-exchange dironiutugrapliy oJji?rni B R N A pu!,.tncwsr. (A) Form B RNA polymerase (2600 enzyme units) from DEAESephadex was subjected to chromatography on phosphocellulose and 1300 enzyme units were recovered. (B) Form B RNA polymerase (1 300 enzyme units) was subjected to chromatography on DEAE-cellulose and 2400 units were recovered. (0 ) RNA polymerase activity; (--) absorbance at 280 nm: ( - - - - - - ) (NH,),SO, concentration

Table 1. Purification table ,for the Jorm A R N A polymerase.? The purification procedure was carried out as described in Materials and Methods. Values for protein after the phosphocellulose stage cannot be accurately determined owing to the presence of addcd bovine serum albumin Stage of purification

RNA polymerase activity

Nucleoli 0.07 M KCI extract 0.12 M KCI extract DEAE-cellulose cnzyme Phosphocellulose A1 Phosphoccllulose A11 DEAE-Scphadex A1 DEAE-Sephadcx A11 Glycerol-gradient A1 Glycerol-gradient A1 I

Yield

Protein

Spccific activity

units

2 3

mg

units;'mg protein

6663

100

47 1

22'7] 2355 4572

69

83

4063

61

1537 2788 956 1470 144 197

2 3 ) 65 42

13 2.25 2.08

'1

22 36 2) 5 3

-

0.15' 0.15"

14 55 313 582 1340 960 1313

Thesc values were dcterinined from absorbance measurements at 280/260 nm and from Coomassie blue staining in polyacrylamidc gels.

separation of the form B enzyme on columns of phosphocellulose and DEAE-cellulose is shown in Fig. 7. The final specific activities of forms AI, A11 and B were 960, 1313 and 2447 units/mg protein respectively. It should be noted that the specific

activity of the B enzyme prepared here From nucleoplasm is about 10-fold lower than that made from nuclei, as described previously [21]. This is possibly due to inactivation during the early stages of the present procedure caused by dilution of nucleoplasm.

33

B. E. H. Coupar and C. J . Chesterton Table 2. Purifi'cution table f o r the form B R N A polymerases The purification procedure was carried out as described in Materials and Methods Stage of purification

R N A polymerase activity

Yield

units Nucleoplasm Sonicated nucleoplasm supt Dialysed (N H,),S04 precipitate DEAE-Sephadex eluate Phosphocellulose eluate DEAE-cellulose eluate

7700 3140 1260 4173 6320 4160

This step is necessary to isolate clean nucleoli. Electrophoresis on native polyacrylamide gels of each enzyme from the peak fraction of the final purification stage shows each to be reasonably homogeneous (Fig. 8). Each of the bands seen co-sediments with the enzyme on a glycerol gradient. The whole final preparation of each enzyme does contain other protein impurities but, as reported below, these are not nucleases. As found previously [21] and by Kedinger etal. [24], form B consists of at least two types of enzyme which can be separated by electrophoresis, form BI migrating more slowly than the form BII. We find the ratio of these types variable from preparation to preparation, and it seems likely that form BI decreases with increasing age of the rat. However, no detailed studies have becn carried out. The enzyme preparations were tested for the presence of DNase and RNase activity, as described in Materials and Methods. Neither was detected in the form A preparations at, and beyond, the DEAESephadex stage. Form B required the DEAE-cellulose step to remove last traces of both nucleases. The basic properties of the enzymes are demonstrated by the results in Table 3. All enzymes are severely inhibited by omission of ATP, GTP, CTP or DNA from the normal reaction mixture. Both form A RNA polymerases show higher activity on nucleolar DNA whereas form B activity is depressed on this template (these results will be analysed further in a subsequent paper). The reverse results are obtained with denatured DNA, thereby agreeing with previous data [18]. None of the enzymes showed affinity for T4 DNA. DNase or actinomycinD treatment of rat liver DNA abolished most of the activity. That the product formed is RNA was demonstrated by its destruction in the presence of either RNase or alkali. Preincubation with rifamycin AF/013 inhibited all activity as expected [15,25]. Whereas form B is inactivated by 1 pg/ml a-amanitin, the form A enzymes are resistant even up to 200 pg/ml of the drug, showing that form C (or 111) RNA polymerases are absent [2,4,26]. The results shown in Table 3 also show that the form A enzymes prefer

100

41 16 54 82 54

Protein

Specific activity

mg

units/mg protein

1575 1000 432 15.4 3.1 1.7

5 3.3 2.9 27 1 2039 2441

Fig. 8. Polyrrcrylamitlr gel c,lectrt)pliorc,.\is of R N A i ~ ( i I ~ i i i e r a . ~ c ~ . ~ . Form A1 (4.1 units), form A11 (7.9 units) and form B (12.2 units) RNA polymerases were subjected to electrophoresis on 4 polyacrylamide gels. Form A1 and form A11 RNA polymerase samples were from glycerol gradient fractions similar to those shown in Fig. 6. The form B sample was from a peak of RNA polymerase activity from a DEAE-cellulose column similar to that shown in Fig. 7 B

M g + ions ot Mn2+ ions and show optimal activity under the low salt incubation conditions. Conclusions

When purifying form A DNA-dependent RNA polymerase from rat liver, it is clear that nucleoli are the best source to use. We show here that a relatively nuclease-free homogeneous preparation of the en-

34

R. E. H. Coupar and C. J. Chesterton: Eukaryotic RNA Polymerases

Table 3. General characteristics of the purified RNA polymerases Apart from the factors under test, all incubations were carried out as described in Materials and Methods for each form RNA polymerase and contained 2.93, 0.64 or 2.33 enzyme units of forms AI, A11 or B respectively. DNase I (80 pg/ml) was preincubated for 10 min at 37°C with the incubation mixture before addition of enzyme. Pancreatic RNase (20 pg in 0.02 ml water) or 0.1 ml of 1 N NaOH was added after 10 min and the incubation continued for a further 20 min. Controls for these contained water in place of RNase or alkali. Actinomycin D was present at 36 pg/ml and a-amanitin as indicated. Enzymes were preincubated with 200 pg/ml rifamycin AFj013 for 5 min at 37 "C. Magnesium and manganese chloride concentrations were 5 mM and 1 mM respectively. Low and high salt indicates the presence of 0.04 M and 0.13 M (NH,),SO, respectively. Normal rat liver DNA was omitted from incubations containing nucleolar, T4, or denatured DNA. Templates were used at 40 pg/ml and denaturation was achieved at 100°C for 15 min. Values shown represent the activity as a percentage of similar normal control incubations Experimental condition

RNA polymerase activity -

.~

form A1

form A11 form B

% Normal incubation minus ATP minus CTP minus GTP minus D N A plus nucleolar DNA plus denatured DNA plus T4 DNA plus DNase plus RNase plus alkali plus actinomycin D plus rifamycin AF/013 plus a-amanitin 1 pg/ml plus a-amanitin 200 pg/ml plus Mn2 , minus Mg2 plus MnZ+ ,plus M$ plus Mg2+,low salt plus M$+,high salt plus Mn2 , minus Mg2 , high salt +

+

+

+

100 2.2

100

100 4.8 1.6 3.1 0.7 63 133 0.5 0.8

-

12.0 4.6 7.4 0.3 181 44 5.5 0.1 4.2 3.3 2.8 4.6 106 88 50 -

16

-

115 71

47

-

54

42

-

1.1 6.9 0.8 138 28 8.0 0.2 3.4 0.2 1.1 0.5 107 91 45

-

1.5" 0.4 0.8 0.8 3.3

-

+

a RNA was ectracted with sodium dodecylsulphate/phenol/ CHCI, and precipitated with ethanol before incubation with RNase at 20 pg/ml.

zymes can be obtained from this sub-nuclear organelle. For most purposes, the enzymes need not be purified further than the DEAE-Sephadex stage since nuclease is absent and preparations are concentrated. Also, severe losses of enzyme occur at the later stages of purification. Form B is best purified from low-salt-extracted nuclei, as we have described previously [21,27]. However, if nucleoplasm must be used, the procedure here

can be adopted as it does yield a nuclease-free and fairly homogeneous product. However, the yield and specific activity of the purified enzyme is relatively low. We warmly thank Dr Peter Butterworth and Dr Trevor Beebee for all our interesting discussions. We are indebted ro Professor L. G. Silvestri of Gruppo Lepetit for the gift of rifamycin AF/013, and to Professor T. Wieland for the gift of a-amanitin. The excellent technical assistance of Stevan Lewis is gratefully acknowledged. This work was supported by the Science Research Council (Grant no. B/RG/4270.8).

REFERENCES 1 . Jacob, S. T. (1973) Progr. Nucleic Acid. Res. Mol. Biol. 13, 93-126. 2. Weimann, R. & Roeder, R. G. (1974) Proc. Nail Acad. Sci. U.S.A. 71, 1790-1794. 3. Kedinger, C., Nuret, P. & Chambon, P. (1971) FEBS Left. I S , 169- 174. 4. Austoker, J. L., Beebee, T. J. C., Chesterton, C. J. & Butterworth, P. H. W. (1974) Cell, 3,227-234. 5. Weiland, T. (1968) Science (Wash. D.C.) 159, 946-952. 6. Widnell, C. C. & Tata, J. R. (1966) Biochim. Eiophys. Acra, 123,478 - 487. 7. Roeder, R. G . & Rutter, W. J. (1969) Nature (Lond.) 224, 234-237. 8. Roeder, R. G. & Rutter, W. J. (1970) Proc. Nail Acad. Sci. U.S.A.65,675-679. 9. Reeder, R. H. & Roeder, R. G. (1972) J. Mol. Eiol. 67, 433441. 10. Yu, F. L. & Feigelson, P. (1972) Proc. Natl Acad. Sci. U.S.A. 69,2833-2837. 11. Gross, K. J. & Pogo, A. 0. (1974) J . Eiol. Chem. 249, 568576. 12. Beebee, T. J. C. & Butterworth, P. H. W. (1974) Eur. J . Biochem. 45,395 - 424. 13. Beebee, T. J. C. & Butterworth, P. H. W. (1974) FEBS Lett. 47,304- 306. 14. Beebee, T. J. C. & Butterworth, P. H. W. (1974) Eur. J . Biochem. 44.1 15 - 122. 15. Butterworth, P. H. W., Cox, R. F. & Chesterton, C. J. (1971) Eur. J. Biochem. 23, 229-241. 16. Gros, F. (1974) FEES Lett. 40, S19-S27. 17. Chesterton, C. J. & Butterworth, P. H. W. (1971) Eur. J . Biochem. 19,232-241. 18. Chesterton, C. J. & Butterworth, P. H. W. (1971) FEBS Lett. 12,301 - 308. 19. Gissinger, F. & Chambon, P. (1972) Eirr. J . Biochem. 28, 277 - 282. 20. Paul, J. & Gilmour, R. S. (1968) J. Mol. Eiol. 34, 305-316. 21 Chesterton, C. J . & Butterworth, P. H. W. (1971) FEBS Letr. 15,181-185. 22 Busch, H. (1967) Metho& Enzymol. 12A, 448-453. 23 Burgess, R. R. (1971) Annu. Rev. Biochem. 40, 711-740. 24. Kedinger, C., Gissinger. F. & Chambon, P. (1974) Eur. J . Biochem. 44,421 -436. 25. Meilhac, M. & Chambon, P. (1973) Eur. J. Biochem. 35, 454- 463. 26. Seifart, K. H., Benecke, B. J. & Juhasz, P. P. (1972) Arch. Biochem. Biophys. 151.519-525.

B. E. H. Coupar and C. J. Chesterton, Department of Biochemistry, King's College London, Strand, London, Great Britain, WC2R 2LS