purification subunit structureof dna-dependent rna polymerase iii

Plant Physiol. (1981) 67, 438-4440032-0889/81/67/0438/07$00.50/0

Purification and Subunit Structure of DNA-dependent RNAPolymerase III from Wheat Germ'

Received for publication July 11, 1980 and in revised form September 11, 1980

JERRY JENDRISAKDepartment of Botany, University of Minnesota, St. Paul, Minnesota 55108

ABSTRACT

A rapid and simple, large-scale method for the purification of DNA-dependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented.The method involves enzyme extraction at low ionic strength, polyethyl-eneimine fractionation, (NH4)2SO4 precipitation, and chromatography onDEAE-Sepharose CL-6B, DEAE-cellulose, and heparin agarose. Milligramquantities of highly purified enzyme can be obtained from kilogram quan-tities of starting material in 2 to 3 days. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis indicates that RNA polymerase III contains 14subunits with molecular weights of: 150,000; 130,000; 94,000, 55,000,38,000; 30,000-, 28,000; 25,000; 24,500, 20,500; 20,000; 19,500;, 17,800, and17,000. Subunit structure comparison of wheat germ RNA polymerases I,II, and III indicates that all three enzymes may contain common subunitswith molecular weights 20,000, 17,800, and 17,000. In addition, RNApolymerases II and III may contain a common subunit with a molecularweight of 25,000, and RNA polymerases I and III may contain a commonsubunit with a molecular weight of 38,000.

Studies with a variety of eukaryotic species indicate that tran-scription of the nuclear genome involves the participation of threeclasses of DNA-dependent RNA polymerases (EC 2.7.7.6) whichcan be distinguished chromatographically, structurally, function-ally, and by differential sensitivity to inhibition by the fungaltoxin a-amanitin (reviewed in refs. 7 and 31). Chromatographyon DEAE-Sephadex is the most effective means for resolving thevarious enzymes in a single chromatographic step (31), and RNApolymerases I, II, and III have thus been detected in wheat (24)and rye (12) embryos by this method.

Results from studies with several plant species indicate thatplant RNA polymerases closely resemble the cognate mammalianenzymes with respect to inhibition by a-amanitin (12, 15, 24, 28).RNA polymerase I is not inhibited by a-amanitin (12, 15, 24),RNA polymerase II is inhibited by low conceuitrations of a-amanitin (50% inhibition at 0.01-0.05 ,ug/ml) (12, 15, 28), andRNA polymerase III is inhibited by relatively high concentrationsof a-amanitin [50%o inhibition of the wheat (24) and rye (12)enzymes by 2.5-10 ,g/ml].

Results from studies with wheat embryos (25), soybean hypo-cotyl nuclei (18), and cultured plant cells (33) indicate, in agree-ment with results obtained with animal systems (31) and yeast (19,32), that RNA polymerase I transcribes the genes for the largerRNAs (18), that RNA polymerase II transcribes the genes form(poly A') RNA (25, 33), and that RNA polymerase III tran-scribes the genes for the low-molecular weight cellular RNAs, i.e.

'This research was supported by Grant GM 24294 from the NationalInstitutes of Health.

5S rRNA and tRNAs (25).Eukaryotic nuclear RNA polymerases are multisubunit en-

zymes with aggregate mol wt of 500,000 to 750,000 (31). Thesubunit structures of RNA polymerases I, II, and III, as deter-mined in animal systems (31) and yeast (19), are distinctly differ-ent. They superficially resemble each other in that they are allcomposed of two high molecular weight subunits (mol wt >100,000), and several small subunits. Most of the subunits aredifferent according to molecular weight. In yeast (19) and mam-malian (31) systems there is evidence which indicates that a fewlow molecular weight subunits are shared in common by all threeenzymes. In plant systems, only RNA polymerase II has beenwell-characterized according to subunit structure (27, 28). This islargely due to the fact that RNA polymerase II is the mostabundant of all three RNA polymerases and is the most amenableto purification. Few studies have been done on the subunit struc-ture of either RNA polymerases I or III from plants, althoughsome preliminary subunit structures have been reported for RNApolymerase I in wheat (23), soybean (17), and cauliflower (15) andfor RNA polymerase III in wheat (40). The results generallyindicate subunit structures similar to those proposed for the cog-nate animal (31) and yeast (19) enzymes.

Here, methodology for the large-scale purification of RNApolymerase III from wheat germ is presented. This procedure hasthe advantage over other RNA polymerase purification scheme inthat large quantities of starting material can be handled to yieldmg quantities of highly purified enzyme. The subunit structurewas analyzed by SDS-PAGE,2 and the results are compared withthose reported by others (40). Significant discrepancies in theproposed subunit structure for wheat RNA polymerase III arediscussed. In addition, evidence is presented for common subunitsin all three RNA polymerases from wheat.

MATERIALS AND METHODS

MATERIALS

Wheat germ was obtained from General Mills, Inc., Vallejo,CA. Heparin was obtained from Gibco. Sepharose 4B and DEAE-Sepharose CL-6B were obtained from Pharmacia. DEAE-cellulose(DE52) was obtained from Whatman. Cyanogen bromide andPEI were obtained from Eastman. Sources ofchemicals for buffersand for SDS-PAGE were as described previously (26, 28). Thefollowing proteins used as molecular weight markers were ob-tained from the indicated sources: BSA (Sigma); myoglobin, f8-lactoglobulin, and a-chymotrypsinogen (Calbiochem); and crea-tine phosphokinase (Boehringer-Mannheim). Wheat germ RNApolymerase I was purified as described by Jendrisak (23, 24).Wheat germ RNA polymerase II was purified according to Jen-

2 Abbreviations: PAGE, polyacrylamide gel electrophoresis; PEI, poly-ethyleneimine; TEB, 0.050 M Tris-HCl (pH 7.9), 1.0 mm EDTA, 0. lo0 (v/v) 2-mercaptoethanol; TEB-EG, TEB + 25% (v/v) ethylene glycol.

438

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RNA POLYMERASE III FROM WHEAT GERM

drisak and Burgess (26). Escherichia coli RNA polymerase waspurified according to Burgess and Jendrisak (6).

BUFFERS

All Tris buffer pH values are reported at 4 C. All bufferscontained 0.050 M Tris-HCl (pH 7.9), 1.0 mM EDTA, and 0.10%o(v/v) 2-mercaptoethanol. Chromatography buffers in additioncontained 25% (v/v) ethylene glycol. The above buffers alsocontained the indicated concentrations of(NH4)2SO4. Buffers wereconveniently prepared from stock solutions of 10 x TEB [0.50 MTris-HCl (pH 7.9); 10 mM EDTA, and 1.0%o (v/v) 2-mercaptoeth-anol], ethylene glycol, and 3.0 M (NH4)2SO4, all of which werestored at room temperature. Glass distilled and deionized H20and the highest grade chemicals were used for all solutions.

COLUMN CHROMATOGRAPHY

Heparin agarose was prepared by the method of Bickle et al.(2) using CNBr-activated Sepharose 4B and was fimally washed ina column with TEB-EG + 0.35 M (NH4)2SO4 until equilibratedwith this buffer. DEAE-Sepharose CL-6B was washed in a columnwith TEB-EG + 0.25 M (NH4)2SO4 until equilibrated with thisbuffer. DEAE-cellulose was washed in a column with TEB-EG+ 0.125 M (NH4)2SO4 until equilibrated with this buffer. In allcases, column height to column diameter ratios were approxi-mately 5, and flow rates were 2 column volumes/h.

OTHER METHODS

RNA polymerase activity was determined by the method ofDynan et al. (11). One unit of enzyme activity is the amountcatalyzing the incorporation of 1 nmol UTP into trichloraceticacid-precipitable form in 15 min at 30 C. Protein was estimatedby the method of Bradford (3) using BSA as a standard. SDS-PAGE (0.75-mm thick slab gels) was carried out according toLaemmli (29): Gels were stained as described previously (6). Saltconcentrations were determined by conductivity measurements(26). PEI, 10%1o (v/v), was prepared for use as described previously(26).

PURIFICATION OF RNA POLYMERASE III

All steps were carried out at 0 to 4 C and all centrifugationswere for 15 min at 10,000 g (Beckman JA-O0 rotor, 10,000 rpm).

1. Preparation of Crude Extract. Wheat germ (1.0 kg) wasblended for 1 min at full speed with 4.0 liters TEB + 0.050 M(NH4)2SO4 in a 1-gallon Waring Blendor. The homogenate wascentrifuged and the resulting supernatant was filtered through onelayer of Miracloth to give fraction 1 (the crude extract).

2. PEI Fractionation. To the crude extract was added 0.10volume 10% (v/v) PEI with stirring. After 15 min additionalstirring, the mixture was centrifuged, and the supernatant wasdecanted and discarded. The precipitate was suspended in 2.0liters TEB + 0.175 M (NH4)2SO4 with the use of a Polytronhomogenizer. It was operated at a speed which resulted in com-plete suspension of the precipitate with minimal foaming. Theresulting suspension was centrifuged to yield fraction 2 in thesupernatant (the PEI eluate).

3. (NH4)2SO4 Precipitation. To the PEI eluate was added solid(NH4)2SO4 in a ratio of 25 g/100 ml, with stirring. After anadditional 15 min stirring, the precipitate was collected by cen-trifugation and the supernatant was discarded. The (NH4)2SO4precipitate was dissolved in sufficient TEB-EG so that the fmal(NH4)2SO4 concentration was 0.25 M. The resulting solution was

clarified by centrifugation to yield fraction 3 [the (NH4)2SO4precipitate].

4. DEAE-Sepharose CL-6B Chromatography. Fraction 3 pro-

tein was applied to a 50-ml column of DEAE-Sepharose CL-6Bequilibrated with TEB-EG + 0.25 M (NH4)2SO4. The column waswashed with the same buffer until no protein could be detected inthe eluate. RNA polymerase III was step eluted from the columnwith TEB-EG + 0.50 M (NH4)2S04. Fractions containing the high-salt step eluted protein were pooled to give fraction 4 (the DEAE-Sepharose CL-6B pool).

5. DEAE-cellulose Chromatography. Fraction 4 protein wasprecipitated by adding 1 volume saturated (NH4)2SO4 solution.After 1 h at 0 C, the precipitate was collected by centrifugationand was dissolved in sufficient TEB-EG so that the (NH4)2SO4concentration was 0.125 M. The resulting solution was applied toa 25-ml DEAE-cellulose column equilibrated with TEB-EG +0.125 M (NH4)2SO4. The column was washed with the same bufferuntil no protein could be detected in the eluate. RNA polymeraseIII does not bind to this column under these conditions and iscollected in the flow-through fractions which were pooled to givefraction 5 (the DEAE-cellulose pool).

6. Heparin Agarose Chromatography. Fraction 5 protein wasadjusted to 0.35 M (NH4)2SO4 with 3.0 M (NH4)2SO4 and wasapplied to a column of heparin agarose equilibrated with TEB-EG + 0.35 M (NH4)2SO4. The column was washed with the samebuffer until no protein could be detected in the eluate. RNApolymerase III was eluted with a linear gradient of (NH4)2SO4from 0.35 to 0.75 M in TEB-EG. Fractions containing RNApolymerase III were pooled to give fraction 6 (the heparin-agarosepool).

CONCENTRATION AND STORAGE OF RNA POLYMERASE III

The pooled RNA polymerase III-containing fractions from theheparin agarose column were dialyzed for 2 days against TEB-EG containing saturated (NH4)2SO4. The resulting precipitate wascollected by centrifugation at 20,000g for 20 min and was dissolvedin a small volume (less than 0.5 ml) ofTEB + 50% (v/v) glycerol.RNA polymerase III was stored frozen at -70 C, where it isindefinitely stable.

RESULTS

A summary of the purification of RNA polymerase III fromwheat germ is presented in Table I. RNA polymerase activity isextracted from wheat germ in a buffer of low ionic strength. RNApolymerase III is purified along with RNA polymerases I and II(24) through the PEI fractionation step, which removes all of thenucleic acids and a large bulk of the protein (26). Subsequent(NH4)2SO4 precipitation results in additional protein purification,removes residual PEI, and affords a significant concentration ofRNA polymerases (26). RNA polymerases then are subjected tochromatography on DEAE-Sepharose CL-6B. Figure 1 shows a

Table I. Summary of Purfi'cation of Wheat Germ RNA Polymerase IIIThe data reported in this table were taken from a single representative

experiment. 1,000 g of tissue was used as starting material. One unit ofactivity is equal to I nmol UMP incorporated in 15 min at 30 C. RNApolymerase III activity could only be accurately measured in later steps inthe purification procedure, as discussed in the text.

Purification Step Volume Protein Activ- Specificity Activity

ml mg units units/mg1. Crude extract 3680 108,6002. PEI eluate 1950 10,1403. (NH4)2SO4 precipitate 150 2,7304. DEAE-Sepharose CL-6B pool 50 1155. DEAE-cellulose pool 61 8.4 35 4.26. Heparin-agarose pool 47 0.73 31 43

439Plant Physiol. Vol. 67, 1981



Plant Physiol. Vol. 67, 1981

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FIG. 1. DEAE-Sepharose CL-6B chromatography of wheat RNA po-

lymerases. Fraction 3 protein in the (NH4)2SO4 precipitate, which containsall three RNA polymerases, was applied to a column of DEAE-SepharoseCL-6B in the presence of TEB-EG + 0.125 M (NH4)2SO4 . The flow-through fractions (2-10) contained protein not binding to the column andnegligible RNA polymerase activity. Fractions 22 to 29 contained proteineluted with TEB-EG + 0.25 M (NH4)2SO4 and only RNA polymerase I as

determined by assaying column fractions in the presence of 0 and 100 ,ug/ml a-amanitin. Fractions 43 to 46 contained protein eluting with TEB-EG+ 0.50 M (NH4)2SO4 which contained largely RNA polymerase II, as

determined by assaying column fractions in the presence of 0 and I ,ug/mla-amanitin. (0), protein; (0), RNA polymerase activity.

representative profile obtained when the protein in the (NH4)2SO4precipitate is applied at 0.125 M (NH4)2SO4 and followed by saltsteps at 0.25 and 0.50 M (NH4)2SO4. No RNA polymerase activityis detected in the flow-through fractions. A small amount ofenzyme activity is eluted with a large quantity of protein at 0.25M (NH4)2SO4. The activity is totally resistant to very high concen-trations of a-amanitin and is classified as RNA polymerase I bythis criterion (24). A large quantity of RNA polymerase activityis eluted with 0.50 M (NH4)2SO4 which is greater than 95%inhibited by 1.0 ,ug/ml a-amanitin and is classified as largely RNApolymerase II (28). When the sample is applied to DEAE-Seph-arose CL-6B at 0.25 M (NH4)2SO4, the a-amanitin-resistant activityis found in the flow-through fractions and only the a-amanitinsensitive activity is found to bind to the column. The latterprotocol is usually used for the purification of RNA polymeraseIII. Precise quantitation of RNA polymerase III activity at theseearly stages in the purification procedure was difficult due to thefact the concentration (activity) of RNA polymerase III is verylow. The presence of relatively high concentrations (activity) ofRNA polymerase II also complicated quantitation ofRNA polym-erase III. It was difficult to distinguish between RNA polymeraseIII and residual RNA polymerase II activity when assays wereperformed in the presence ofvarious concentrations ofa-amanitin.Consequently, no entries were made in Table I for RNA polym-erase III activity at early stages of purification. The fimal yield ofenzyme obtained in the end for a number of preparations wasquite reproducible.

I found that the protein eluted from the DEAE-Sepharose CL-6B column with 0.50 M (NH4)2SO4 contained significant quantitiesof RNA polymerase III after subsequent DEAE-cellulose chro-matography (Fig. 2). Protein was applied in the presence of 0.125M (NH4)2SO4 to this column, followed by a wash with a buffer ofthe same ionic composition. The column then was step eluted withbuffer containing 0.25 M (NH4)2SO4. Figure 2A shows the elutionpattern of protein (Amo) from this column. Figure 2B shows thepolypeptide pattern of protein in column fractions as analyzed bySDS PAGE. Samples of purified wheat germ RNA polymerasesII and III were run on this same gel to aid in the detection of

FIG. 2. DEAE-cellulose chromatography of wheat RNA polymerasesII and III. Fraction 4 protein in the DEAE-Sepharose CL-6B pool whichcontains RNA polymerases II and III, was applied to a column of DEAE-cellulose in the presence of TEB-EG + 0.125 M (NH4)2SO4. The protein(A2we) elution profile is presented (A) along with an SDS, 7.5% polyacryl-amide gel containing aliquots from the indicated column fractions andsamples of purified RNA polymerases II and III (B). Fractions 6 to 19contained protein in the flow-through and fractions 21 to 30 containedprotein eluting with TEB-EG + 0.25 M (NH4)2SO4. Numibers to the leftand right of the gel indicate the molecular weights x 10-3 of putative RNApolymerase III and II subunits, respectively.

RNA polymerase II and III subunits across the elution profile.The 150,000, 130,000, 94,000, 55,000, and 38,000 mol wt putativeRNA polymerase III subunits (see below) can be clearly seen inthe flow-through fractions, and the 220,000 and 140,000 mol wtsubunits of wheat germ RNA polymerase 11 (27) can be seen inthe 0.25 M (NH4)2SO4 step eluted protein peak. Cross-contami-nation of RNA polymerases II and III after DEAE-cellulosechromatography appears to be negligible from an inspection ofthis chromatographic profile. The activity in the flow-throughfractions was 50%'o inhibited by 2.5 ,ug/ml a-amanitin and theactivity of the 0.25 M (NH4)2SO4 peak fractions was 50% inhibitedby 0.05 ,tg/ml a-amanitin (data not shown).

Final purification of RNA polymerase III was achieved byheparin-agarose chromatography (Fig. 3). It can be seen that amajor peak of RNA polymerase activity elutes at approximately0.50 M (NH4)2SO4 from this column, which coincides with a peakof protein (A2so) (Fig. 3A). Aliquots of column fractions weresubjected to SDS-PAGE and several polypeptides can be seen tobe associated with RNA polymerase III activity across the columnprofile (Fig. 3B). Apparent contaminants removed at this step canbe seen in the flow-through fractions and also eluting just aheadof the RNA polymerase III peak. The RNA polymerase activityin these fractions appears to be minor traces ofRNA polymeraseII since the activity is inhibited completely by very low concentra-tions of a-amanitin (data not shown).

440

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FIG. 3. Heparin agarose chromatography of wheat RNA polymeraseIII. Fraction 5 protein in the pooled DEAE-cellulose flow-through frac-tions was applied to a column of heparin agarose in the presence of TEB-EG + 0.35 M (NH4)2SO4. RNA polymerase III was eluted with a linear(NH4)2SO4 gradient from 0.35 to 0.75 M in TEG-EG. The elution profileis presented (A) indicating A280 (... ), salt concentrations (0), and RNApolymerase activity (0) in fractions; along with an SDS, 15% polyacryl-amide gel containing aliquots from the indicated column fractions (B).Lines to the right of the gel indicate polypeptides which could be seen on

the gel to coelute with RNA polymerase III activity.

Subunit Structural Analysis of RNA Polymerase III. To deter-mine the polypeptide subunit composition of wheat germ RNApolymerase III, the heparin-agarose-purified enzyme was sub-jected to SDS-PAGE in gels containing 5, 7.5 and 15% polyacryl-amide (Fig. 4). In 5 and 7.5% gels (Fig. 4, A and B), three highmolecular weight polypeptides (mol wt, 150,000, 130,000, and94,000) can be seen. These molecular weights were estimated froma comparison of polypeptide electrophoretic mobilities with thoseof E. coli RNA polymerase subunits (30). The putative RNApolymerase III high-molecular weight subunits are distinctivelydifferent in size from the two high molecular weight subunits inwheat RNA polymerase II (mol wt, 220,000 and 140,000) and thetwo high molecular weight subunits in wheat RNA polymerase I(mol wt, 200,000 and 125,000).Low molecular weight polypeptides associated with RNA po-

lymerase III were examined on 15% gels (Fig. 4, C and D). RNApolymerase III was examined along with wheat RNA polymerasesI and II as well as with various proteins which served as molecularweight markers. Fourteen polypeptides can be seen in RNApolymerase III under these conditions of electrophoresis. In ad-dition to the subunits with mol wts of 150,000, 130,000, and 94,000,polypeptides with mol wts of 55,000, 38,000, 30,000, 28,000, 25,000,24,500, 20,500, 20,000, 19,500, 17,800, and 17,000 are associatedwith RNA polymerase III after heparin agarose chromatography.These are indicated in the densitometric scan of stained 5 and 15%

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FIG. 4. SDS-PAGE of wheat RNA polymerase III. A, RNA polymer-ease III was analyzed with wheat RNA polymerases I and II and with E.coli (EC) RNA polymerase on a 5% gel; B, RNA polymerase III was

analyzed with RNA polymerase II and with various protein markers (M),including E. coli RNA polymerase subunits and BSA, on a 7.5% gel; C,RNA polymerease III was analyzed with RNA polymerase II and withvarious protein markers (M) on a 15% gel; D, RNA polymerases I, II, andIII were analyzed on a 15% gel. Numbers indicate polypeptide molecularweights x lo0.

gels (Fig. 5). The molecular weight calibration curves resultingfrom electrophoresis of molecular weight markers on 5 and 15%polyacrylamide slab gels are shown in Figure 6 where the migrat-ing positions of RNA polymerase III associated polypeptides are

indicated with arrows. The tentative subunit structure of RNApolymerase III is summarized in Table II along with subunitstructures proposed for wheat RNA polymerases I (23) and II(27).Common Subunits in Wheat RNA Polymerases I, II, and III.

SDS-PAGE of wheat RNA polymerases I, II, and III on 15%polyacrylamide gels (Fig. 4D) indicates that polypeptides withmol wts of 20,000, 17,800, and 17,000 are found in all threeenzymes. In addition, a polypeptide with a mol wt of 25,000 isseen in RNA polymerases II and III, and a polypeptide with a

mol wt of 38,000 is seen in RNA polymerases I and III. Theseresults are also indicated in Table II.

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MOBILITY

FIG. 5. Densitometric scans of RNA polymerase III-associated poly-peptides in stained SDS polyacrylamide gels. High molecular weightpolypeptides were separated on a 5% gel and the low molecular weightpolypeptides were separated on a 15% gel. Slab gels were scanned at 550nm using a Beckman 5230 recording spectrophotometer equipped with a

gel scanning attachment. One-half ,ug protein was applied to the 5% geland 2.0 ,ug were applied to the 15% gel. Numbers above the A peaksindicate polypeptide molecular weights x 10-3.

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FIG. 6. Molecular weight estimation of putative wheat RNA polym-erase III subunits by SDS-PAGE. Standard curves were constructed frommigration positions ofproteins ofknown molecular weights in Tris-glycine-buffered (29), SDS, 5% (0), and 15% (0) polyacrylamide gels. Proteinstandards included: a and b, the ,B and ,B' subunits of E. coli RNApolymerase (mol wt, 165,000 and 155,000, respectively) (5); c, the a subunitof E. coli RNA polymerase (mol wt, 90,000) (30); d, BSA (mol wt, 66,296)(4); e, creatine phosphokinase (mol wt, 40,000) (10); f, a-chymotrypsinogin(mol wt, 25,700) (20); g, ,B-lactoglobulin (mol wt, 18,400) (39); and h,myoglobin, (mol wt, 17,200) (42). Arrows indicate the migration positionsof wheat RNA polymerase III-associated polypeptides.

DISCUSSION

I have shown that RNA polymerase III may be readily purifiedin mg quantities from kg quantities of wheat germ. The enzymewas extracted by homogenization of tissue in low ionic strengthbuffer. This contrasts with the high salt [0.50 M (NH4)2SO41procedures used by others (40) for the extraction ofRNA polym-erase III from plant tissue. Low salt extraction procedures are

increasingly being used for the purification of RNA polymerasesT, II, and III from eukaryotic organisms (1, 9, 24, 35, 36), as itappears that a portion of, or in many cases the bulk of, the RNApolymerase activity is not tightly associated with nuclei or chro-matin.

After enzyme extraction, many RNA polymerase purificationprocedures often include a high-speed centrifugation step to re-

move ribosomes and chromatin, etc., and sometimes overnightdialysis against large quantities of buffer. No dialysis, ultracen-trifugation, or other time-consuming and scale-limiting steps are

used in this procedure. Several recent improvements in the meth-

Table II. Summary of Subunit Structures of Wheat Germ RNAPolymerases I, II, and III

Subunit Molecular Weights of Following Polymerases

Ia Ilb IIIC (III)dX 10-3

200 220 150 (155)140 130 (132)125

94 (91)

50 55 (53)4240

| 38 38 (37)32

30 (33)

27 28 (31)25 25 (28)

24 24.5 (26)21 20.5

20 20 2019.5 (16)

17.8 17.8 17.817.0 17.0 17.0

16.316.0

a Data from Jendrisak (23).b Data from Jendrisak and Burgess (27).c Data from the study presented here.d Data from Teissere et al. (40).'Numbers in boxes indicate polypeptides which appear to be shared

between any two or all three RNA polymerases.

odology for the purification of RNA polymerases were includedin this procedure. These include PEI fractionation (6, 26, 43),DEAE-Sepharose CL-6B chromatography (34), and heparin-aga-rose chromatography (37). The advantages of these steps havebeen discussed by others previously but can be briefly summa-rized. RNA polymerases are precipitated, along with total nucleicacids and a fraction of the cellular protein, from crude extractswith PEI at low ionic strength. RNA polymerases are extractedfrom the PEI precipitate with high ionic strength buffer, whereasnucleic acids remain precipitated along with a fraction of theprotein. Since nucleic acids and the bulk of the protein areremoved batchwise with the use of a centrifuge, large quantities ofstarting material can be processed and RNA polymerase solutionsare reduced to volumes and protein concentrations more suitablefor subsequent column chromatographic steps (26). PEI fraction-ation has been successfully used for the purification of RNApolymerases T, TT, and III from Acanthamoeba castellanii (9, 35,36), RNA polymerases II and III from yeast (1), RNA polymeraseII from calf thymus (21) and Physarum polycephalum (34), RNApolymerase I from Aspergillus nidulans (38), RNA polymerase IIfrom Agaricus bisporus (41), and RNA polymerase II from avariety of plant species (16, 28). PEI fractionation appears to begenerally applicable for purification of RNA polymerases T, TT,and III from all eukaryotic organisms.DEAE-Sepharose CL-6B was found to bind wheat RNA polym-

erases more tightly and to have a much higher capacity for RNApolymerases than did other commercially available DEAE-substi-tuted matrices. This allowed the use of much smaller columnswhich resulted in shorter chromatographic times and much higherRNA polymerase concentrations after elution. These results agreewith those of Smith and Braun (34) who mentioned these advan-tages of DEAE-Sepharose CL-6B for the purification of P. poly-

442 JENDRISAK

_.-




cephalum RNA polymerase II. The tighter binding and highercapacity is most likely due to the high-molecular weight sievingrange of Sepharose CL-6B which the high-molecular weight RNApolymerases can penetrate.

Final purification of RNA polymerase III was achieved byheparin-agarose chromatography. RNA polymerases bind to hep-arin agarose very strongly even at fairly high ionic strength,whereas most other cellular proteins are not retained. Heparinagarose has recently been used for the purification of RNApolymerase I and III from A. castellanhi (35, 36), RNA polymeraseII from P. polycephalum (34), RNA polymerase III from wheat(40). E. coli RNA polymerase (37), RNA polymerase III fromcultured animal cells (22), RNA polymerase II from Drosophilamelanogaster (14), and bacteriophage N4 RNA polymerase (13).Heparin agarose also seems to be generally applicable for thepurification of RNA polymerases, and other enzymes involved innucleic acid metabolism (2) from bacterial and eukaryotic cells.

Teissere et al. (40) have reported that polypeptides with thefollowing mol wt are associated with wheat RNA polymerase III:155,000, 132,000, 91,000, (70,000, 66,000), 53,000, 37,000, 33,000,31,000, 28,000, 26,000, and 16,000. They suggest that the 70,000and 66,000 mol wt polypeptides may not be subunits, but contam-inants, because the stoichiometry of these polypeptides is variableduring purification. Results obtained here would also suggest thatthese polypeptides are contaminants because they are not foundin RNA polymerase III purified as detailed here. For the mostpart, there is excellent agreement concerning the size and numberof polypeptides found to be associated with RNA polymerase IIIin these two studies (Table II). Only minor differences in molec-ular weights have been assigned to various polypeptides. Onemajor difference, however, concerns the fact that only one poly-peptide was observed at a mol wt of 16,000 by Tessiere et al. (40),whereas five polypeptides in this molecular weight range wereobserved here (20,500, 20,000, 19,500, 17,8000, and 17,000). Fail-ure to detect these polypeptides may have been due to insufficientquantities ofRNA polymerase III applied to SDS-polyacrylamidegels.

Fourteen polypeptides are associated with purified RNA polym-erase III from wheat germ as indicated here. This agrees well withthe complexity of the subunit structures proposed for A. castellanii[15-17 subunits (35)], yeast [11-13 subunits (1, 19)], and animal[10 subunits (22, 31)] class III RNA polymerases. The size rangeof these polypeptides in all species examined, including higherplants, appears to be fairly conserved.

Earlier studies on the subunit structure of wheat-germ RNApolymerase III (40) have been extended to include a direct com-parison of the putative subunit structures of wheat RNA polym-erases I, II, and III on SDS-polyacrylamide gels. Results indicatethat three low molecular weight polypeptides (mol wt, 20,000,17,800, and 17,000) may be shared in common by all three wheatenzymes. On additional polypeptide may be shared in commononly by RNA polymerases II and III (mol wt, 25,000, and onepolypeptide may be shared in common by RNA polymerases Iand III (mol wt, 38,000). A comparison of these results with thosereported for the well-characterized A. castellanii enzymes (8) in-dicates some striking similarities. Three low molecular weightsubunits (mol wt, 22,000, 15,000, and 13,300) were found to beshared by all three A. castellanii enzymes. There may be homol-ogous to the three low molecular weight polypeptides found inthe wheat enzymes. However, three other subunits were found tobe shared by A. castellanii RNA polymerases I and III (mol wt,39,000,27,000, and 17,500) as opposed to one subunit in the wheatenzymes (mol wt, 38,000). No subunits were found to be shared incommon by A. castellanii RNA polymerases II and III, but it issuggested here that one polypeptide may be shared in common bywheat polymerases II and III (mol wt, 25,000).

Examination of densitometric scans derived from stained SDS

polyacrylamide gels (Fig. 5) suggests that several of the putativeRNA polymerase III polypeptide subunits are present in nonin-tegral amounts. This observation has been made concerning thesubunit structures of RNA polymerases from a variety of eukar-yotic species (7-9, 31). This may be due to differential dye(Coomassie blue) binding or it may reflect variations in thenumber of polypeptides which remain bound to the RNA polym-erase during purification. No variation in the stoichiometrics ofany of the polypeptides tentatively defined as RNA polymeraseIII subunits was observed in many different enzyme preparations.

Clearly, more definitive studies are required, and are in progress,concerning the demonstration of common subunits in plant RNApolymerases I, II, and III. These include two-dimensional (e.g.isoelectric focusing-SDS) polyacrylamide gel analysis and poly-peptide mapping studies. The roles of these putative commonsubunits in eukaryotic RNA polymerases are presently unclear. Ithas been suggested that they may be required for a commonfunction intrinsic to all three enzymes (8). The ability to obtainlarge quantities of highly purified RNA polymerase III and othernuclear RNA polymerases from wheat germ (24) should facilitatefurther studies on the nuclear RNA polymerases and the signifi-cance of common subunits.

Acknowledgment-The excellent technical assistance of Ruby J. Larson is grate-fully acknowledged.

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