Purification and Subunit Structure of Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase III from the Mouse Plasmacytoma, MOPC 315’”

(Received for publication, July 16, 1975)

VIRGIL E. F. SKLAR$ AND ROBERT G. ROEDER§

From the Department of Biological Chemistry, Division of Biology and Biomedical Sciences, Washington University, St. Louis, Missouri 63110

Class III DNA-dependent RNA polymerases were purified from the mouse plasmacytoma, MOPC 315. RNA polymerases III, and III, 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 chromato- graphically homogeneous Enzymes III, and III, 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 subunit compositions of chromatographically purified RNA polymerases III, and III, were ana- lyzed by polyacrylamide gel electrophoresis under denaturing conditions. RNA polymerase III A contained subunits with molecular weights of 155,000 (IIIa), 138,000 (IIIb), 89,000 (IIIc), 70,000 (IIId), 53,000 (IIIel), 49,000 (IIIe2), 41,000 (IIIfj, 32,000 (IIIg,J, 29,000 (IIIh), and 19,000 (1111). RNA polymerase III R subunits were identical with those of Enzyme III, except for the replacement of subunit IIIg. with a slightly larger subunit IIIg B (M, = 33,000). Molar ratios were close to unity for all subunits except for 1111, which was present in stoichiometric excess, yielding a composite molecular weight of approximately 695,000. Analysis of purified RNA polymerases III A and III R by polyacrylamide gel electrophoresis under nonde- naturing conditions revealed, in each instance, two major protein bands. Subsequent analyses of the two electrophoretic forms of Enzyme III A failed to reveal any structural differences since each form contained subunits IIIa to i in the same proportions as found in the unfractionated phosphocellulose enzyme.

The present data have been used to estimate the cellular concentrations of RNA polymerase III mole- cules. MOPC 315 cells have approximately 3 x 10’ molecules/cell, while the concentrations in other cell types are estimated to be as much as 50-fold lower. However, the specific activities and subunit composi- tions of the enzymes purified from mouse plasmacytoma cells and from normal tissues appear similar. Thus, fluctuations in the cellular levels of RNA polymerase III activity may, in part, result from changes in the cellular concentration of RNA polymerase III molecules. The data are discussed in terms of the regulation of transfer RNA and 5 S RNA synthesis and cell growth rate by RNA polymerase III.

DNA-dependent RNA polymerase III represents one of the three principal classes of nuclear RNA polymerase found in

polymerase III usually accounts for a small proportion of the

eukaryotic cells (1). RNA polymerase III was originally distin- total RNA polymerase activity and has been detected, using

guished from RNA polymerases I and II on the basis of its appropriate analytical methods, in all tissues examined (2).

distinct catalytic properties and its elution from DEAE- Chromatographically heterogeneous forms of these enzymes h

Sephadex at high salt concentrations (l), features which now ave been described in mouse plasmacytomas (2), mouse liver

appear common to most or all class III enzymes (l-13). RNA and spleen tissues (2), human peripheral lymphocytes (3), calf thymus (2), rat liver (9), and Xenopus laeuis liver tissue.’ In

* This work was supported in part by National Institutes of Health contrast, only one class III enzyme has been detected in lower

Grant l-ROl-CA16640 and by National Science Foundation Grant eukaryotes and this enzyme appears resistant to high concen-

BMS 74-24657. trations of cu-amanitin (10-13). There has been no report of + Predoctoral Fellow supported by the National Institutes of Health physical, structural, or functional differences among the het-

Training Grant 5 TO1 GM-1311. 8 Recipient of Research Career Development Award 1.K04-

erogeneous class III enzymes.

GM-70661 from the National Institutes of Health. ’ R. G. Roeder, unpublished observations.

1064

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Subunit Structure of Mouse Plasmacytoma RNA Polymerase III 1065

In the mouse plasmacytoma, class III RNA polymerases have been shown to synthesize tRNA and 5 S RNA species (14). The cellular levels of solubilized RNA polymerase III activity vary among different cell types and in the same cell type under different physiological conditions (2-4) and may therefore regulate directly the cellular rate of tRNA and -5 S RNA synthesis. Determination of the specific mechanism(s) ac- counting for these variations in RNA polymerase III activity might provide insights into the regulation of tRNA and 5 S RNA synthesis and cell growth rates.

To investigate these problems, we have chosen the mouse plasmacytoma, MOPC 315,2 a rapidly growing malignant cell. These cells contain high levels of RNA polymerase III (2), probably reflecting a high level of tRNA and 5 S RNA synthesis characteristic of a rapid rate of cellular proliferation (15). This paper reports the purification and subunit structures of the class III enzymes from MOPC 315 cells, which have permitted a structural comparison of homologous class I, II, and III RNA polymerases (16). Evidence is presented that the heterogene- ous class III enzymes, designated III, and III., have minor differences in their physical properties and subunit composi- tions. These studies also provide evidence that fluctuations in the levels of RNA polymerase III activity may, in part, be mediated via changes in enzyme concentration.

EXPERIMENTAL PROCEDURES

Cells

MOPC 315 solid tumors were obtained as described previously (2).

Biochemicals

Unlabeled nucleoside triphosphates were obtained from P-L Bio- chemicals; [3H]UTP from New England Nuclear; crystalline bovine serum albumin from Pentex; acrylamide, bisacrylamide, and tetra- methylethylenediamine (TEMED) from both Eastman and Bio-Rad Laboratories; a-amanitin from Henley and Co.; calf thymus DNA type I, Nonidet P-40, PMSF, and iPr,P-F from Sigma; and [d(A-T)], from Miles.

Ion Exchange Resins

DEAE-cellulose (Whatman DE52) and phosphocellulose (Whatman P-11) obtained from Reeve Angel were prepared as described previ- ously (17, 18). DEAE-Sephadex (A-25) and CM-Sephadex (C-25) purchased from Pharmacia were prepared as described previously (2). The resins were stored at O-4” and prior to use were deaerated under vacuum.

Conductiuity, DNA, and Protein Measurements

As previously described (2), salt concentrations were measured with a Radiometer conductivity meter (type CDM 2e) following 200.fold dilutions of the samples with water. DNA was measured according to the method of Burton (19). Protein was measured according to the method of Lowry et al. (20), after the samples were precipitated with trichloroacetic acid.

Assay for RNA Polymerase

Assays were performed in a final volume of 25 111 as described previously (3). Calf thymus DNA or [d(A-T)], was used as template at a final concentration of 100 or 50 pg/ml, respectively. One unit of activity represents incorporation of 1 pmol of UMP into RNA in 20 min under the previously described conditions with calf thymus DNA (unless otherwise specified). For a given series of experiments the same template batch was always used because RNA polymerase activity varied among different template preparations.

‘The abbreviations used are: MOPC 315, mineral oil plasmacytoma 315; TEMED, tetramethylethylenediamine; PMSF, phenylmethylsul. fonylfluoride: iPr,P-F, diisopropylfluorophosphate; Trizma, 2.amino. 2-hydroxymethyl-l,%propanediol.

Purification Methods

RNA Polymerase Solubilitation-RNA polymerase was solubilized from whole cells by a modification of a previously published procedure (2). MOPC 315 tumors (100 g) were first homogenized (15 strokes) in 200 ml of Buffer A (0.05 M Tris-HCl (pH 7.9, 23’), 25% (v/v) glycerol, 0.1 mM EDTA, 0.5 rnM dithioerythritol, 5 mM M&l,, 1 mM PMSF, and 1 rnM iPr,P-F) and passed through a double-layer of cheesecloth. The suspension was adjusted to 0.32 M ammonium sulfate and the viscous solutions were sonicated as described previously (2). This solution was centrifuged for 20 min at 18,000 rpm in a Sorvall type SS-34 rotor. The supernatants were centrifuged for 120 min at 50,000 rpm in a Spinco type Ti-60 rotor. The resultant 0.32 M ammonium sulfate supernatant fractions (Fl) were diluted to 0.1 M ammonium sulfate with Buffer A and aggregated chromatin was pelleted by centrifugation for 50 min at 50,000 rpm. To the 0.1 M ammonium sulfate supernatant fractions (F2), solid ammonium sulfate (Mann enzyme grade) was added to saturating levels (0.42 g/ml). The precipitate was pelleted by centrifu- gation for 70 min at 35,000 rpm in a Spinco type 42 rotor. The ammonium sulfate pellets were resuspended by addition of Buffer B (Buffer A minus MgCI, and iPr,P-F) and homogenization and then diluted to 0.19 M ammonium sulfate by the addition of Buffer B (F3). This suspension was then centrifuged for 50 min at 50,000 rpm. The resulting 0.19 M ammonium sulfate supernatant fractions (F4) were pooled and stored at -80”. All procedures were performed at O-4”.

DEAE-cellulose Chromatography-Fraction F4 was subsequently thawed and diluted with Buffer B to a final salt concentration of 0.06 M

ammonium sulfate. This sample was loaded onto a DEAE-cellulose column equilibrated with Buffer B containing 0.05 M ammonium sulfate (4 to 6 mg of protein/ml of bed volume). The resin was washed with 2 column volumes of the equilibration buffer and eluted with a linear gradient of 0.05 to 0.25 M ammonium sulfate in Buffer B. The total gradient size was equivalent to 4 column volumes and fractions equivalent to 2.5% of the gradient volume were collected. Following this, the remainder of the adsorbed activity was eluted with Buffer B containing 0.5 M ammonium sulfate. Appropriate column fractions containing class I plus III RNA polymerases were combined and assayed for activity and protein content.

DEAE-Sephadex Chromatography-The combined fractions from DEAE-cellulose chromatography were directly loaded onto a DEAE- Sephadex column equilibrated with Buffer B containing 0.1 M ammo- nium sulfate (1 to 1.5 mg of protein/ml of bed volume). The resin was washed with 1 column volume of equilibration buffer and 1 column volume of Buffer B containing 0.05 M ammonium sulfate and eluted with a linear gradient of 0.05 to 0.5 M ammonium sulfate in Buffer B. The gradient size was 3 column volumes and fractions corresponding to 2.5% of the gradient volume were collected. Fractions containing RNA polymerase III, were combined and assayed for enzyme activity and protein content. Bovine serum albumin was then added (0.5 mg/ml) to help stabilize enzyme activity and the solution stored at -80”. Similarly, fractions containing RNA polymerase III,, were combined, bovine serum albumin was added, and the solution stored at 80”. The following procedures were used to purify further the chromatographi- tally separated RNA polymerases III ,, and III “.

CM-Sephadex Chromatography-The combined fractions from DEAE-Sephadex chromatography (i.e. containing either III, or III,) were thawed and dialyzed against 10 volumes of Buffer B to a final concentration of 0.04 M ammonium sulfate. The dialyzed preparation was loaded onto a CM-Sephadex column equilibrated with Buffer C (Buffer B plus 0.5 mg/ml of bovine serum albumin) containing 0.05 M ammonium sulfate. Excluding the carrier bovine serum albumin, 0.5 to 1.0 mg of protein was loaded/ml of bed volume. The resin was washed with 2 column volumes of Buffer C containing 0.03 M ammonium sulfate. The enzyme was eluted with a linear gradient of 0.03 to 0.25 M ammonium sulfate in Buffer C. The gradient size was 3 column volumes and fractions equivalent to 3% of the gradient volume were collected. During the loading and washing of the column and during gradient elution of the column the flow rates were, respectively, 0.06 and 0.033 ml/min/ml of bed volume. Fractions containing RNA polymerase III were pooled and reassayed for enzyme activity with calf thymus DNA.

First Phosphocellulose Chromatography-The CM-Sephadex en- zyme was diluted to 0.08 M ammonium sulfate by the addition of Buffer C. This preparation was loaded onto a phosphocellulose column equilibrated with Buffer C containing 0.05 M ammonium sulfate (50,000 to 100,000 units of activity/ml of bed volume). The resin was washed with the equilibration buffer and the enzyme was then

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1066 Subunit Structure of Mouse Ph +macytoma RNA Polymerase II1

concentrated by a step elution with a solution containing 0.0.5 M Tris-HCI (pH 7.9), 20’7 (v/v) glycerol, 0.1 rn~ EDTA, 0.5 rn~ dithioerythritol, 1 mg/ml of bovine serum albumin, 1 tn~ PMSF, and 0.25 M ammonium sulfate. Fractions of 0.29 ml were collected at a flow rate of 0.5 ml/min and assayed for activity with calf thymus DNA.

Sucrose Density Gradients-The peak activity fractions from the first phosphocellulose column were individually layered directly onto 5 to 20’? sucrose density gradients (4.4 ml) which were prepared and then used exactly as described previously (17). Unless otherwise specified, fraction volumes of 0.22 ml were collected and assayed for activity with calf thymus DNA. Although the purification effected by this procedure IS not evident from the data in Table II, omission of this step resulted in an increased background of low molecular weight polypeptides ( <80,000) in the final phosphocellulose enzyme prepara- tion ( below).3

Second Phosphocellulose Chromatography-The peak activity frac- tions from the sucrose gradients were individually loaded onto a second phosphocellulose column equilibrated with Buffer B containing 0.05 M ammonium sulfate (100,000 to 150,000 units of activity/ml of bed volume). The resin was washed with Buffer B containing 0.05 M ammonium sulfate and eluted with a 10 column volume linear gradient of 0.05 to 0.5 M ammonium sulfate in Buffer B. Fraction volumes equivalent to 30; of the gradient volume were collected and individ- “ally assayed for RNA polymerase activity and protein content. These Individual fractions were then stored at 80”.

Po~,wcry:ylamide Gel Electrophoresis-Phosphocellulose gradient fractions containing RNA polymerases III, or III,, were subjected to electrophoresis on polyacrylamide gels in cylindrical tubes under both denaturmg and nondenaturing conditions as described previously (17).

The denaturing sodium dodecyl sulfate gel system is a modification of Laemmli’s procedure (21). These gels were fixed in 12’% (w/v) trichloroacetlc acid and 50% (v/v) ethanol for 5 to 10 hours at 50” and stained in 10% (v/v) acetic acid, 50% (v/v) ethanol, and 0.14 (w/v) Coomassie brilliant blue for 10 to 20 hours at 50”. Gels were destained and stamed bands were scanned with a Gilford linear transport device as described previously (17).

Electrophoresis under nondenaturing conditions was carried out on polyacrylamide gels which were constructed according to the method of Maize1 (22). Fresh dithioerythritol and bromphenol blue were added to each sample to final concentrations of 20 rn~ and O.O05Lt, respectively. This solution was then layered over a 1.3ml polyacrylamide gel (6.j x 0.5 cm) containing 5% acrylamide, 0.13% bisacrylamide, 25% (v/v) glycerol. O.:i75 M Tris-HCI (pH KY), 0.15 rn~ dithioerythritol, 0.06% (v/v) TEMED, and (~.08~1 (w/v) ammonium persulfate. The gel tubes were then filled with electrode buffer (5 rn~ Trizma base and 38 rn~ glycine). Samples were subjected to electrophoresis at 1 mA/gel until the dye had stacked and entered the gel. and then the current was increased to 1.5 mA/gel until the dye front reached the bottom of the gel. These gels were either stained for protein or sliced. In the latter case, slices were subjected to electrophoresis under denaturing condi- tions as described previously (17).

In addition, class III RSA polymerases were subjected to electropho- resis under denaturing conditions on a high resolution polyacrylamide gel slab (25 x 9 x 0.1 cm) using a procedure similar to that described bs Studier (23). The resolving gel, 24 cm in length. contained a linear 6 to ll’r acrylamide gradient. A 3Y acrylamide stacking gel, 1 cm in length. was polymerized above the gradient resolving gel. Electropho- resis of the samples through the stacking gel was carried out at 110 volts (constant voltage) for 90 min and the voltage was then raised to 150 volts until the tracking dye had completely run through the resolvmg gel (i.e. approximately 12 hours total). The gel slab was fixed for 3 ho&s at 25” and stained for 4 hours at 25”, using the solutions described above. The gel slab was destained with several washes of lo’% (v/v) acetic acid and then photographed after removal of the stacking gel. Unless otherwise stated, all reagents used for poly- acrylamide gel electrophoresis were those obtained from Bio-Rad Laboratories.

RESULTS

Solubilization, Chromatography, and Sucrose Gradient Sedi- mentation-Solubilized extract (F4), containing class I, II,

and III RNA polymerases, was obtained as described under “Experimental Procedures.” As summarized in the upper part of Table I, these prechromatographic procedures remove 65%’

3V. E. F. Sklar, unpublished observations.

TABLE I

This table summarizes the activity yields of total (i.e. class I 1 II ,

III) and class III RNA polymerases from 100 g of MOIY 315 tumor.

Whole cells (containing approximately 25 g of protein and 600 mg 01

DNA) were homogenized, and soluhilized enzyme Fractions Fl, F2, F3, and F4 were obtained and assayed for RNA polymerase activitv and

protein content as described under “Experimental Procedures.” ‘I otal

activity was measured in the absence of a-amanitin. I’rior IO IIF:AIS- Sephadex chromatography, the levels of class III RNA pulymerases

(i.e. III, + Ill,,) were estimated as the amount of activity which was

inhibited only by high concentrations of a-amanitin. Class III activity

was not measured in Fractions Fl, F2, and F3. Data for DEAE-cel- lulose chromatography were calculated from Fig. 1 and except for

“Total Activity” represent only the fractions insensitive to low

concentrations of a-amanitin (i.e. containing class I plus class III

enzymes). Data for DEAE-Sephadex chromatography were calculated

from Fig. 2 and refer to the fractions containing RNA polymerases III, and III,. Assuming that the amounts of RNA polymerases III, and III,,

in the crude whole cell extract (containing 25 g of’ protein) are

equivalent to those determined after resolution of the enzyme on

DEAE-Sephadex (Table II). the specific activities of enzymes III,4 and

III, appear to he 0.014 and 0.0058 units/& of protein, respectively, in

the crude extract. The data reported here and in Table II were taken from one representative experiment.

ml w

Fl 0.32 M ammonium 250 9,200

sulfate superna-

tant fraction

F2 0.1 M ammonium 710 6,100

sulfate superna-

tant fraction

F3 resuspended am- 290 3,900

monium sulfate

precipitate

F4 0.19 M ammonium 285 3,300

sulfate superna-

tant traction

DEAE~cellulose ?I50 1,100

DEAF,-Sephadex 700 70

1,850,000

“.m~,ooo

%.400,000 4’0.000 0. 1

4.:~00.000 ,FJ50,000 0.5

495,000 7.1

of the contaminating protein in Fraction Fl, with no detectable loss in total RNA polymerase activity. The apparent increase in activity during these initial purification steps may he due to removal of inhibitory proteins.

RNA polymerase Fraction F4 was subjected to the chromato- graphic and sedimentation procedures described under “Exper- imental Procedures.” Chromatography of Fraction F4 on DEAE-cellulose reveals two major peaks of activity (Fig. 1).

The activity eluting at 0.07 to 0.13 M ammonium sulfate represents a mixture of class I and III RNA polymerases as determined by its insensitivity to low concentrations (0.5 /*g/ml) of a-amanitin (2) and by its elution behavior on

DEAE-Sephadex (see below). The activity eluting from DEAE-cellulose at 0.16 to 0.30 M ammonium sulfate was completely sensitive to low concentrations of a-amanitin and therefore represents exclusively RNA polymerase II (2).

The peak DEAF,-cellulose fractions, insensitive to low levels of a-amanitin, were combined and subjected to DEAE- Sephadex chromatography (Fig. 2). This procedure resolves RNA polymerase I and the individual class III RNA polymer-

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Subunit Structure of Mouse Plasmacytoma RNA Polymerase III 1067

FIG. 1. DEAE-cellulose chromatography of whole cell RNA polym- erase. Solubilized RNA polymerase Fraction F4 containing 3.3 g of protein and approximately 2.4 x lo8 units of activity was obtained from 100 g of tumor and chromatographed on a 600-ml column (13.5 x 7.5 cm) containing DEAE-cellulose. Fraction volumes of 50 ml (Fractions 1 to 32) and 100 ml (Fractions 33 to 40) were collected at a flow rate of 15 ml/min and assayed for RNA polymerase activity. The activity recovered in gradient Fractions 1 to 21 (2,130,OOO units) was completely resistant to 0.5 fig/ml of a-amanitin, while~the activity in Fractions 22 to 40 (2,155.OOO units) was completely sensitive to 0.50 &ml of Lu-amanitin. No activity was detected in the breakthrough fractions. Fractions 8 to 18 were combined and assayed for protein content. The data summarized in Table I (except for “Total Activity”) are based on values for these 11 fractions. Activity was measured with calf thymus DNA in the absence (04) or presence (0- - -0) of 0.5 rig/ml of cY-amanitin; -, ammonium sulfate concentration; ., absorbance at 280 nm.

FIG. 2. DEAE-Sephadex chromatography. Combined fractions from DEAE-cellulose chromatography (Fig. 1) containing 1.1 g of protem and approximately 2 x lOa units of activity were chromato- graphed on a 900.ml column (20.5 x 7.5 cm) containing DEAE- Sephadex. Fraction volumes of 580 ml (Fractions 1 to 4) and 70 ml (Fractions 5 to 40) were collected at a flow rate of 16 ml/min and assayed for RNA polymerase activity. Due to the high concentration of ammonium sulfate (0.1 M) in the combined DEAE-cellulose fractions, approximately 800,000 units of activity were not adsorbed to the DEAE-Sephadex column. This activity was recovered in Fractions 1 to 4, was insensitive to 400 pg/ml of a-amamtin, and displayed a [d(A-T)]JDNA activity ratio of 0.9. The activity recovered in gradient Fractions 15 to 19 (670,000 units) was also completely resistant to 400 &ml of ol-amanitin and displayed a [d(A-T)],JDNA activity ratio of 1.3. The activity recovered m gradient Fractions 27 to 36 (495,000 units) was completely sensitive to 400 fig/ml of cu-amanitin and displayed a [d(A-T)],JDNA activity ratio of 11.1. The data summa- rized in Table I are based on values for Fractions 27 to 36. Fractions 27 to 31, containing RNA polymerase III,, were combined, assayed for protein content, and stored at -80”. Fractions 32 to 35, containing RNA polymerase III,, were similarly combined. Activity was measured in the presence of 0.5 Kg/ml of cz-amanitin with [d(A-T)]” (0-O) and with calf thymus DNA (O---O); -, ammonium sulfate con- centration; ., protein (mg/ml).

ases, which are further identified on the basis of amanitin sensitivity and relative activity using native DNA uersus [d(A-T)]” as templates (see below). Due to the high concentra- tion (0.1 M) of ammonium sulfate in the input sample, the majority of the enzyme I activity was not adsorbed (see legend

to Fig. 2). As described previously (2), MOPC 315 cells contain two chromatographically distinct forms of RNA polymerase III, designated III,, and IIIH. As summarized in the lower part of Table I, the DEAE-cellulose and the DEAE-Sephadex chro- matographic steps yield an approximately 70-fold purification

with greater than 90% recovery of class III activity. The following procedures were used to purify further the

chromatographically separated RNA polymerases III, and III,,. Since these two enzymes display similar behavior on CM- Sephadex and phosphocellulose chromatography, as well as on sucrose gradient sedimentation in the presence of 0.08 M

ammonium sulfate, 3 only the purification of RNA polymerase III, will be described.

DEAE-Sephadex fractions containing 349,000 units of RNA polymerase III, activity (Fig. 2) were combined and subjected to chromatography on a CM-Sephadex column (12 x 2.5 cm) as described under “Experimental Procedures.“.Less than 5% of the input activity appeared in the breakthrough fractions. The activity which bound to the column was eluted in a single symmetrical peak at an ammonium sulfate concentration of 0.10 M. A total of 160,000 units of activity were recovered. The peak fractions (containing 135,000 units) were pooled and loaded onto a phosphocellulose column (1.8 x 1.2 cm) as described under “Experimental Procedures” (see “First Phos- phocellulose Chromatography”). All of the activity was ad- sorbed to the column and eluted in a single sharp peak with a maximal enzyme concentration (peak tube) of 120,000 units/ ml. A total of 105,000 units of activity were recovered. Those fractions which contained enzyme concentrations in excess of 30,000 units/ml were subjected individually to sucrose gradient sedimentation at an ammonium sulfate concentration of 0.08 M, as described under “Experimental Procedures.” A total of 95,000 units were loaded onto the sucrose gradient and the apparent yield of activity in this step was 103%. The sedimen- tation profile was similar to that observed in experiments described below. The final purification step was adsorption of the enzyme from the peak sucrose gradient fractions to a second phosphocellulose column and elution with a linear salt gradient (Fig. 3).

Overall Purification and Recovery-The purification of the individual class III enzymes after DEAE-Sephadex chromatog- raphy is summarized in Table II. Specific activities shown for the final phosphocellulose enzymes are the average from all enzyme-containing fractions. Specific activities measured across the peak phosphocellulose fractions averaged 310 units/ rg of protein with calf thymus DNA and 5,450 units/fig of protein with [d(A-T)],. The apparent specific activities of RNA polymerases IIIA and IIIB in the crude cellular extract are, respectively, 0.014 and 0.0058 units/pg of protein with calf thymus DNA as template (see legend to Table I). Thus RNA polymerases III, and III, are purified approximately 22,000 and 53,000-fold, respectively, relative to whole cell extracts.

As summarized in Table II, 14% of the initial RNA polymer- ase III, activity and 15% of the initial RNA polymerase III, activity, measured with calf thymus DNA, were recovered in the final phosphocellulose fractions. When activity was mea- sured with [d(A-T)],, 20% recovery of both Enzymes III, and III, was observed. However, as detailed in the legends to Figs. 1

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1068 Subunit Structure of Mouse Plasmacytoma RNA Polymerase III

to 3 and in the text, not all of the activity recovered at each step was collected for subsequent purification procedures. Had no activity been discarded at each of these stages, an overall yield of 27% or 39’ir should have been attained, as measured with calf thymus DNA or [d(A-T)],, respectively. These recoveries of activity were obtained by minimizing the number

75 I h

FIG. 3. Second phosphocellulose chromatography (linear gradient elution). RNA polymerase III, fractions from the sucrose density gradients (see text) containing 74,000 units of activity were chromato- graphed on a O.&ml column (1.0 x 0.8 cm) of phosphocellulose. Fraction volumes of 0.17 ml were collected at a flow rate of 0.17 ml/min and assayed for RNA polymerase activity and protein content. All of the activity adsorbed to the column. Averaged data for Fractions 8 to 16 are reported in Table II. These fractions were stored at -80”. O---O, Activity with [d(A-T)],; 0- - -0, activity with calf thymus DNA; A-A, protein (fig/ml); -, ammonium sulfate concentra- tion.

TABLE II

Purification summary

This table summarizes the activity yields of RNA polymerases III, and III,,, which were resolved on DEAE-Sephadex chromatography. The initial purification procedures are summarized in Table I. Data for the chromatographic and sucrose gradient sedimentation steps were calculated from Figs. 2 and 3 and from data presented in the text. The activity indicated was determined with calf thymus DNA and repre- sents total activity recovered in each step. Due to the inclusion of bovine serum albumin during CM-Sephadex and first phosphocel- lulose chromatography, as well as sucrose gradient sedimentation, these fractions were not assayed for protein content. The specific activity listed for second phosphocellulose enzymes is an average computed from all fractions containing activity. The specific activities measured across the peak phosphocellulose Fractions 9 to 13 (Fig. 3) were approximately constant, averaging 310 units/pg of protein with native calf thymus DNA and 5450 units/pg of protein with [d(A-T)],.

Fractmn Jik Protein Activity Specific Re- activity covery

ml M Lmrts unltsl %

&%n

RNA polymerase III, DEAE-Sephadex 420 51,000 349,000 6.8 100 CM-Sephadex 40 160,000 46

First phosphocellulose 2.3 105,000 30

Sucrose density gradient 5.5 98,300 28

Second phosphocellulose 1.5 178 48,000 270 14

RNA polymerase III, DEAE-Sephadex 280 19,000 146,000 7.7 100 CM-Sephadex 30 99,000 68

First phosphocellulose 1.2 53,000 36

Sucrose density gradient 5.0 46,000 32

Second phosphocellulose 1.5 83 21,500 260 15

and degree of dilutions of enzyme solutions, by the inclusion of bovine serum albumin in the buffers used for CM-Sephadex and the first phosphocellulose chromatography, and by the addition of bovine serum albumin and Nonidet P-40 to sucrose density gradients (17).

Properties-RNA polymerases III, and III, have many similar properties which distinguish them from the correspond- ing class I and class II enzymes. These include (a) biphasic salt activation profiles with native DNA templates (2); (b) distinct chromatographic behavior on DEAE-cellulose (elution at low ionic strength) uersus DEAE-Sephadex (elution at high ionic strength) (Figs. 1 and 2); (c) sensitivity to high concentrations of Lu-amanitin (50% inhibition at 20 pg/ml) (2); and (d) increased activity (ll- to 1Bfold) with [d(A-T)], as template, relative to native DNA (Figs. 2 and 3).

Thus far only minor differences in the properties of RNA polymerase III, and III, have been detected. As shown above, the enzymes show distinct chromatographic properties on DEAE-Sephadex (Fig. 2). In addition, RNA polymerase III, can be distinguished from RNA polymerase III. by sucrose gradient sedimentation at intermediate ionic strengths (Fig. 4). In the presence of 0.125 M ammonium sulfate, Enzyme III, sediments as a single peak of activity, while Enzyme III, sediments as a double peak of activity (Fig. 4, Panels B and E, respectively). Similar results were observed at 0.1 M

ammonium sulfate.$ At higher concentrations of ammonium

TOP --

Frocf~on No Bo‘lom

FIG. 4. Sucrose gradient sedimentation at different ionic strengths. Samples of RNA polymerases III A (Panels A to C) and III B (Panels D to F) were subjected to sucrose density gradient sedimentation as described under “Experimental Procedures.” In order to better visual- ize the data in the various experiments the activity in each fraction of a given gradient is plotted as a per cent of the activity present in the fraction containing the highest level of enzyme activity (designated 100%). For each experiment the concentration of ammonium sulfate (M), fraction volume collected (ml), and units of activity in the peak tube were, respectively, 0.02,0.13, and 1,000 (PanelA); 0.125,0.22, and 67,100 (Panel B); 0.40, 0.11, and 1,150 (Panel C); 0.05, 0.13, and 450 (Panel D); 0.125, 0.22, and 19,800 (Panel E); and 0.40, 0.11, and 850 (Panel F). Arrows in Panels B and C denote the sedimentation position of Escherichia coli core RNA polymerase in parallel gradients contain- ing 0.125 and 0.40 M ammonium sulfate, respectively. Activity was measured with [d(A-T)]” and in each case greater than 70% recovery of activity was observed. The sedimentation profiles shown are from several experiments, not all of which were done in parallel. These results are representative of at least three separate experiments performed at each salt concentration. From the relationship (MJ M,)*” = S,/S, (36) and a molecular weight of 400,006 (37) for E. coli core RNA polymerase, the molecular weight of RNA polymerase III activity appears to be approximately 650,000 (data from Panels B and Cl.

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Subunit Structure of Mouse Plasmacytoma RNA Polymerase III 1069

FIG. 5. Subunit pattern of RNA po- lymerase III, according to position in the phosphocellulose column gradient. RNA polymerase III, (48,000 units of activity) was purified as described in this report. Equivalent volumes of Frac- tions 9 to 16 (containing 450 to 2,300 units of activity) from the second phos- phocellulose chromatography (Fig. 3) were subjected to electrophoresis in 10% polyacrylamide gels containing sodium dodecyl sulfate as described under “Experimental Procedures.”

sulfate both enzymes sediment as single peaks of activity as shown in Panels C and F (Fig. 4). However, at concentrations of ammonium sulfate lower than 0.1 M (Fig. 4, Panels A and D), both enzymes display heterogeneous peaks of activity. These data are consistent with the idea that Enzyme III, aggregates at both low and intermediate ionic strengths, whereas Enzyme III, does so only at low ionic strengths. Although these enzymes have been purified by chromatography on two cation exchange columns and on two strong anion exchange columns prior to sedimentation on sucrose gradients, the possibility remains that a contaminating substance (e.g. a nucleic acid) may be responsible for the characteristic sedi- mentation properties of RNA polymerases III ,, and III B.

Polyacrylamide Gel Electrophoresis under Denaturing Con- ditions-Individual phosphocellulose gradient fractions, con- taining RNA polymerase III, activity, were subjected to electrophoresis in the presence of sodium dodecyl sulfate (Fig. 5). The 10 polypeptides designated IIIa,b,c,d,el,e2,f,g,h, and i, in order of decreasing molecular weight, are regarded as putative subunits based on the following observations. First, the mass of each of these polypeptides is directly proportional to the enzyme activity present in each phosphocellulose gradient fraction (Fig. 5). In contrast, this relationship does not hold for the additional polypeptides which are apparent in the various gradient fractions, with the possible exception of the 24,000- and 22,000-dalton polypeptides which migrate between

IIIh and IIIi (see also below). Second, the unlabeled polypep- tides in Fig. 8 are not consistently detected in various enzyme preparations and account for less than 3% of the total protein present in peak activity fractions. Third, polypeptides IIIa to h are present in approximately equimolar amounts in each gradient fraction. Although an accurate molar ratio determina- tion could not be obtained for subunit IIIi, this band was observed in stoichiometric excess and may thus represent more than one polypeptide (see below). The molecular weights and molar ratios of RNA polymerase III, subunits are summarized in the second and fourth columns of Table III, respectively.’

‘Under the conditions of electrophoresis used previously (cylindrical gels, Eastman reagents) (16), a polypeptide designated IIIe was present tn a molar ratio of greater than unity (relative to subunit IIIb) and two polypeptides designated IIIfl and IIIf2 were each present in molar ratios less than unity. tinder the conditions of electrophoresis used in Fig. 5 (cylindrical gels, Bio-Rad reagents), polypeptide IIIe is resolved into two components (IIIel and IIIe2) each present in molar ratios of unity (Table III), whereas polypeptides IIIfl and IIIf2 (16) appear to migrate as a single component (III0 which is present in a molar ratio of

TABLE III

Subunit structures Class III RNA polymerase subunit compositions were examined by

polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with enzymes obtained from phosphocellulose gradient frac- tions and with the electrophoretic forms of III, observed on non- denaturing polyacrylamide gels. Molecular weights were averaged from several preparations and varied from the values shown by less than 5% in the case of subunits IIIa and IIIb and by less than 106 in the case of subunits 111~ to i. Molar ratios were normalized to subunit IIIb. Molar ratios for the indicated subunits, measured across phosphocellulose gradient fractions, were fairly constant, varying less than 20% from the average values shown (Fig. 5). Molar ratios for electrophoretic forms III,-1 and III,-2 were obtained from Fig. 7. In some instances (see Fig. 7) additional staining material was apparent in the region between subunit IIIi and the dye front. Thus, molar ratio determinations for subunit IIIi are not as accurate as for the remaining subunits. The electrophoretic forms of Enzyme III, displayed a heterogeneous shoul- der of staining material which migrated slightly faster than subunit IIK. This material was not included in molar ratio determinations for subunit IIIf and has not been further examined. In addition, the indicated molar ratios must be regarded as approximate since the staining intensity of individual proteins may not always be propor- tional to molecular weights (cf. Ref. 26).

Molecular weight Molar ratios

Subunit Phospho- Phospho- Phospho- Electro- Electro- CE!llUlOS~ cellulose CdUlOSe phoretic phoretic

III, III,, III, III,-1 III,-2

i 138,000 155,000 155,000 138,000 1.0 1.0 1.1 1.0 1.1 1.0

Ii 89,000 70,000 89,000 70,000 1.1 1.1 0.9 1.0 1.0 1.0 el 53,000 53,000 1.0 1.1 1.3 e2 49,000 49,000 1.2 1.0 1.0 f 41,000 41,000 1.2 0.9 0.8

hk” 32,000 33,000 0.8 0.9 0.9 h 29,000 29,000 1.1 1.1 0.9 i” 19,000 19,000 l-3 l-3 l-3

a See text for a discussion of the heterogeneity of subunit i.

approximately unity (Table III). However, heterogeneity in polypep- tide IIIf has also been observed in the present studies, when the enzyme is subjected to electrophoresis in a high resolution polyacrylamide gel slab (Fig. 8). The molar ratios of the individual heterogeneous IIIf polypeptides vary in different enzyme preparations, but their sum is approximately unity. The basis for the heterogeneity in subunit IIIf is not known.

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1070 Subunit Structure of Mouse Plasmacytoma RNA Polymerase III

Polyacrylamide Gel Electrophoresis under Nondenaturing Conditions-Highly purified RNA polymerase III, (phos- phocellulose gradient fraction) was analyzed by polyacrylam- ide gel electrophoresis under nondenaturing conditions. When these gels were stained for protein, two major bands (desig- nated III,-1 and III,-2) were routinely observed, and one minor diffuse band was occasionally detected (Fig. 6). Greater than 95% of the protein stain was associated with these bands. The migration of these bands and their relative intensities were somewhat variable in different experiments, yielding two patterns as shown in Fig. 6 (compare Gels 1 and 2). The cause of this variability is not clear, but samples containing high concentrations of enzyme seemed to yield the pattern shown in Gel 1 (Fig. 6), while less concentrated samples yielded the pattern shown in Gel 2 (Fig. 6). However, other factors such as minor differences in the salt concentration of the samples may contribute to this variability. No activity measurements (see Ref. 17) were attempted on the electrophoretically separated protein bands.

Polyacrylamide Gel Electrophoresis under Denaturing Con- ditions Following Electrophoresis under Nondenaturing Condi- tions-The subunit compositions of electrophoretic forms III,-1 and III,-2 have been determined. An unstained poly- acrylamide gel, containing 6 times more sample protein than Gel 1 (Fig. 6), was divided into l-mm wide slices and the protein in the individual slices was subjected to electrophoresis under denaturing conditions as described previously (17). Panel A in Fig. 7 shows the subunit composition of the phos- phocellulose enzyme prior to electrophoresis under nonde- naturing conditions. Panels B and C in Fig. 7 show the poly- peptide compositions of electrophoretic forms III,-1 and III A-2, respectively. The subunit compositions of electro- phoretic forms III,-1 and III,-2 were identical and, except for a shoulder of staining material migrating slightly faster than subunit IIIf, they were the same as that of the phospho- cellulose enzyme. Subunit molecular weights and molar ratios are summarized in Table III.

I 2 3

FIG. 6. Polyacrylamide gel electrophoresis under nondenaturing conditions. Samples of RNA polymerases III A and III k from the second phosphocellulose column fractions were subjected to electrophoresis on 5% polyacrylamide gels under nondenaturing conditions as described under “Experimental Procedures.” Gel 1, electrophorests of RNA polymerase III, (900 units); Gel 2, electrophoresis of RNA polymerase III,, (350 umts); and Gel 3, electrophoresis of RNA polymerase III,, (400 units). Polyacrylamide Gels 2 and 3 were run identmally and in parallel.

The enzyme preparation analyzed in this experiment (Fig. 7) also appeared to contain two polypeptide components between polypeptides IIIh and IIIi (cf. Fig. 5). Although the polypeptide bands were diffuse (Panels B and C), these two components appeared to remain associated with electrophoretic forms III,-1 and III,-2. As noted above, however, these components are not readily detected in all RNA polymerase III preparations.

A small amount of protein was recovered from slices corre- sponding to the minor diffuse band in Gel 1 of Fig. 6. With the probable exception of polypeptides IIId and IIIg*, this material contained all the subunits present in the phosphocellulose

Electrophoret!c Form Ul,-1

&U

a (C)

b d

C

I::i;

Electrophoret,c Form Q.2

el ez

1

,j+&---@l -1 2 4

cm 6

FIG. 7. Polyacrylamide gel electrophoresis under denaturing condi- tions following electrophoresis under nondenaturing conditions. Chro- matographically purified RNA polymerase III, (5300 units) was subjected to electrophoresis under conditions identical with those in Fig. 6. This gel was run in parallel with Gel 1 in Fig. 6. After electrophoresis, the gel was sliced and each slice was subsequently subjected to electrophoresis on 10% polyacrylamide ‘gels containing sodium dodecyl sulfate as described previously (17). Due to the large amount of protein used, bands III,-1 and III,-2 were somewhat broadened. Nevertheless, two peaks of protein, corresponding in migration position to the two electrophoretic forms of III,,, were clearly separated by a gel slice containing negligible amounts of protein.’ Panel A portrays the characteristic subunit pattern for RNA polymer- ase III,, prior to electrophoresis under nondenaturing conditions (see Fig. 5). Panels B and C portray characteristic subunit patterns for the two major bands, designated III,-1 and III,-2, respectively, in Fig. 6. These results were obtained from three polyacrylamide gels which were run in parallel. Enzyme subunits, the molecular weights and molar ratios of which are summarized in Table III, are labeled a to i. Full scale absorbances at 550 nm t-1 were (a), 2.4; (61, 1.2; and (cl, 0.6.

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Subunit Structure of Mouse Plasmacytoma RNA Polymerase III 1071

enzyme8 and may thus represent a partially dissociated RNA polymerase III molecule.

Electrophoretic Comparison of RNA Polymerases IIIA and III,--Electrophoresis of RNA polymerase III” under non- denaturing conditions revealed two major protein bands simi- lar to the results obtained with Enzyme III, (Fig. 6). The subunit compositions of electrophoretic forms IIIR-1 and III,-2 were not investigated.

Electrophoresis of individual phosphocellulose gradient frac- tions containing RNA polymerase III, activity revealed a subunit pattern similar to the one obtained with Enzyme III,.3 To resolve minor differences in the subunit compositions between RNA polymerases III, and IIIs, these enzymes were subjected to electrophoresis individually and in combination on a 25-cm polyacrylamide gel slab under denaturing condi- tions (Fig. 8). These data clearly illustrate that RNA polymer- ases III, and III, differ only in one subunit. Except for a 32,000 dalton subunit IIIgA which is unique to Enzyme III, and a 33,000 dalton subunit IIIgH which is unique to Enzyme IIIB, the

FIG. 8. High resolution polyacrylamide gel electrophoresis of RNA polymerases III, and III, under denaturing conditions. Chromato- graphically purified samples were subjected to electrophoresis in a 25-cm polyacrylamide gelslab containing a 6 to 11% linear acrylamide gradient as described under “Experimental Procedures.” Left, Enzyme III, (1800 units); center, Enzyme III, (1800 units) plus Enzyme III, (1300 units) combined prior to denaturation; right, Enzyme III, (1306 units). Enzyme subunits, the molecular weights rof which are summa- rized in Table III, are labeled a to i (see text).

subunit molecular weights in Enzyme IIIH appear identical with those in Enzyme III, (Table III). The molar ratios of the subunits in Enzyme III” are similar to those for the analogous subunits in Enzyme III,.3 The electrophoretic system used here (Fig. 8) resolves polypeptide IIIf (Fig. 5) into two components as observed previously (Ref. 16, see also Footnote 4). This electrophoretic system also resolves subunit IIIi into several polypeptides. Although subunit IIIi appears to be present in stoichiometric excess after electrophoresis under nondenatur- ing conditions (Fig. 7), it is not clear which of the low molecular weight polypeptides observed in Fig. 8 remain associated with the enzyme under these conditions.

DISCUSSION

Purification, Structure, and Heterogeneity of RNA Polymer- ases ZZZ* and III,-Two chromatographic forms of RNA polym- erase III are present in the mouse plasmacytoma MOPC 315. Previous studies have shown the presence of RNA polymerase III activity in both cytoplasmic and nuclear fractions following cellular disruption and fractionation (2, 5, 8, 9). We have, therefore, purified the MOPC class III enzymes from whole cells in order to study the total cellular population of these molecules. RNA polymerases III, and III, were resolved by chromatography on DEAE-Sephadex and purified by ion exchange chromatography and sucrose gradient sedimenta- tion. Relative to whole cell extracts the overall purifications were 22,000- and 53,000-fold, respectively, for enzymes III, and III,.

Chromatographically homogeneous RNA polymerases III, and IIIR each contains at least 10 putative subunits desig- nated IIIa,b,c,d,el,e2,f,g,h, and i. That these RNA polymerase III-associated polypeptides represent enzyme subunits is sug- gested by the following observations: (a) the ratio of the amount of each polypeptide to the amount of enzyme activity is approximately constant for individual phosphocellulose gradi- ent fractions; (b) the molar ratios of these polypeptides are approximately unity, with the exception of polypeptide IIIi which is present in a higher but constant molar ratio; (c) the molecular weight of RNA polymerase III calculated from the molecular weights and molar ratios of the individual polypep- tides (695,000) is compatible with that estimated from sucrose gradient sedimentation (650,000); (d) polypeptides IIIa to i co-sediment with RNA polymerase III activity upon sucrose density gradient sedimentation and they remain associated with the major protein bands when RNA polymerase III is subjected to electrophoresis under nondenaturing conditions; and (e) the murine and amphibian class III enzymes contain analogous polypeptides of the same or similar size (16), even though these enzymes are from grossly different cell types.

The subunit compositions of RNA polymerases III, and III, are very similar, differing only in subunit IIIg which is slightly smaller in Enzyme III, (IIIg,-32,000 daltons) than in Enzyme III, (IIIgR-33,000 daltons). As reported previously (2), no evidence for interconversion of these enzyme forms could be found since they maintain their distinctive properties upon rechromatography on DEAE-Sephadex. The presence of serine protease inhibitors (i.e. PMSF and iPr,P-F) during enzyme isolation and purification did not alter the subunit patterns of the purified enzymes.3 The general similarity between the subunit structures of Enzymes III, and III, correlates with the similarities in their catalytic properties and a-amanitin sensi- tivities. However, the minor structural difference between subunits IIIg, and IIIg, may be responsible for differences in

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1072 Subunit Structure of Mouse Plasmacytoma RNA Polymerase III

the sedimentation properties and DEAE-Sephadex elution positions of Enzymes III, and III,, and might also reflect functional differences.

Chromatographically purified RNA polymerases III, and III, were each resolved into two electrophoretic forms. The subunit compositions of the electrophoretic forms of RNA polymerase III, (III,-1 and III,-2) were indistinguishable. Therefore, the

electrophoretic forms of RNA polymerase III may differ pri- marily in the charge of a specific subunit(s), which would not be detected by electrophoresis in the presence of sodium

dodecyl sulfate. Alternatively, these electrophoretic forms could reflect different states of aggregation of the enzyme or other minor structural differences not detectable in the present analytical systems.

Alterations in RNA Polymerase Activity-Class III RNA polymerases have been shown to synthesize tRNA and 5 S RNA in mouse plasmacytoma cell nuclei (14). These enzymes have also been shown to synthesize several distinct, low

molecular weight viral RNAs in adenovirus P-infected KB cells5 (24) which, like plasmacytoma cells, contain two chroma- tographic forms of RNA polymerase III (25). These observations and the structural studies presented here suggest the possibility of functional differences between RNA polymerases III, and

III,,. Although the basis and significance of the distinct elec- trophoretic forms of each class III enzyme remain unclear, electrophoretic variants might represent distinct functional states or regulatory modifications of a single enzyme.

Previous studies have shown both increased rates of tRNA and 5 S RNA synthesis (15) and increased cellular levels of solubilized RNA polymerase III activity in more rapidly growing cell types (2, 3, see introduction to the text). In an attempt to distinguish whether increased enzyme activities result from RNA polymerase modifications or from increased

enzyme concentrations, the purified class III RNA polymerases from several cell types have been compared. The intrinsic

specific activity of the MOPC class III enzymes (Table II) is similar to that observed for RNA polymerase III from both Xenopus laeuis oocytes’ and from the posterior silk gland of Bombyx mori3 and is comparable to the specific activities re-

ported for other purified eukaryotic RNA polymerases (17, 18, 26-29). Furthermore, the subunit compositions of the murine and the amphibian class III enzymes are remarkably similar (16). Thus, as reported previously for RNA polymerase I (17), the variable levels of RNA polymerase III activity in different cell types (2) may reflect primarily variations in the cellular concentrations of RNA polymerase III.

From the cellular levels of RNA polymerase III activity (2) and from the specific activities of the purified enzymes it can be estimated that plasmacytoma cells contain approximately 3 x 10’ molecules of RNA polymerase III. Assuming a combined value of 3,000 to 17,000 genes coding for 5 S and tRNAs in mammalian cells (30-32) and that each active gene could

accommodate approximately two RNA polymerases (cf. Ref. 33), it appears that plasmacytoma cells contain sufficient class III enzyme molecules to saturate these genes. In contrast, less active cell types such as liver cells (2) or peripheral lympho-

cytes (3) contain up to 50-fold less RNA polymerase III than plasmacytoma cells and the enzymes may be limiting in these situations.

These considerations suggest a coarse level of regulation of

the cellular rates of tRNA and 5 S RNA synthesis via

’ R. Weinmann, unpublished observations.

alterations of cellular Enzyme III concentrations. Such altera- tions in enzyme levels may, however, reflect long term adaptive

changes and do not rule out the possible involvement of other enzyme regulatory factors, which may have escaped detection by the nonspecific RNA polymerase assay conditions used. Furthermore, since distinct tRNA (34) and 5 S RNA (35) genes appear to be differentially transcribed, additional components which regulate enzyme activity may be anticipated. The complex subunit structure of the class III enzymes suggests the possibility of RNA polymerase III interactions with a variety of distinct cellular components. These interactions may serve to regulate the activity of these enzymes and to integrate this regulation with other cellular processes. Furthermore, the class III enzymes may be regulated independently of the class I and II enzymes since each enzyme class is comprised in large part of distinct polypeptides (16).

1. 2.

3.

4. 5.

6. I. 8.

9.

10.

11. 12.

13.

14.

15. 16.

17.

18.

19. 20.

21. 22.

23. 24.

25.

26.

27.

28.

29.

30.

31.

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V E Sklar and R G Roederacid polymerase III from the mouse plasmacytoma, MOPC 315.

Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic

1976, 251:1064-1073.J. Biol. Chem. 

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