Purification of the Three Nuclear RNA Polymerases from ... - Duke

Vol. 260, No. 30, Issue of December 25, pp. 16169-16173 1985 Printed in L k A .

THEJOURNAL OF BIOLOGICAL CHEMISTRY

0 1985 by The American Society of Biological Chemists, Inc.

Purification of the Three NuclearRNA Polymerases fromNeurospora erama* (Received for publication, April 12, 1985)

Daniele ArmaleoS and SamsonR. Gross From the Department of Biochemistry, Division of Genetics, Duke University, Durham, North Carolina 27710

Nuclear RNA polymerases I, 11, and I11 from Neu- ative agarose electrophoresis, a procedure developed specifirospora crassa have been purified3,000-, 1,500-, and cally forthis purpose. The results of this analysis suggest that lO,OOO-fold, respectively, by a procedure that mini- the 700-kDa complex, whichis found in the nucleus, may be mizes proteolysis of the220-kDa subunit of polymer- functionally associated with polymerase I1 (Armaleo and ase 11. The Neurospora enzymes resemble, in polypep- Gross, 1985). tide composition, the corresponding polymerases isolated from other eukaryotes. The220-kDa subunit of MATERIALS AND METHODS~ Neurospora polymerase I1 cross-reacts with antisera directed against the 220-kDa subunits oftype I1 polymRESULTS AND DISCUSSION erases from Drosophila and wheat germ. However, the Purification Procedure-A variety of methods used in the proteolyzed 180-kDa derivative ofthe Neurospora 220-kDa subunit fails to cross-react with the heterol- isolation of RNA polymerases fromdifferent organisms were ogous antisera, suggesting that the region removed bycombined in a single procedure for the purification of the proteolysis contains or contributes to structural fea- three nuclear RNA polymerases from N. crassa. RNA polymtures of the enzyme that have been highly conserved erases I, 11, and I11 were identified on the basis of the order during evolution. A 700-kDa complex of 12 polypep- with which they eluted from DEm-Sephadex (Roeder, 1976), tides was found associatedwith polymerase I1 during the similarities of the subunit compositions to those of the purification. The complex was eventually separated correspondingpolymerases fromyeast, and thefact that Neufromtheenzyme,and its properties suggest that it rospora polymerase 11, despite its high resistance to a-amanmight be associated with polymerase I1 in the nucleus. itin (see below), was moresensitive to thetoxin than polymWe describe two additional examples of polypeptides erases I and 111. associated in variable amounts with Neurospora poThe innovations introduced to minimize proteolysis inlymerase 11. volved addition of phenylmethylsulfonylfluoride to thegrowing culture shortly before harvesting and carrying out the initial extraction steps at low temperature (-15 “ C ) in 20% dimethyl sulfoxide. Aninitial Polyimine P fractionation (JenEukaryotic nuclear RNA polymerases have been purified drisak and Burgess, 1975; Smith and Braun, 1978), followed from a variety of sources including several species of fungi by ion filtration chromatography (Hager et al., 1977), rapidly (Lewis and Burgess, 1982). Previous work on the enzymes removed large amounts of protein and nucleic acid.Heparinfrom Neurospora crassa, however, was limited to the chro- agarose chromatography was used here for the fiist time in matographic separation of multiple activity peaks in crude the simultaneous purification of all three RNA polymerases extracts (Timberlake and Turian, 1974; Tellez de Inon et al., and provided an efficient separation of polymerase I11 from 1974). While much is known about genetic regulation in the other two enzymes. The separation of polymerase I from Neurospora, little is known about the structural and regula- I1 was accomplished bychromatography on DEAE-Sephadex tory properties of the proteins involved intranscription (Tyler (Roeder, 1976) whichalso provided a method for separating et al., 1984). It seemed appropriate, then, to purify the RNA polymerase I11 from contaminating chromatin. A 700-kDa polymerases of Neurospora for an analysis of promoter selec- complex of 12 polypeptides, associated with polymerase 11, tivity and factors affecting rates of transcription. appeared responsible forthe multiple peak elution pattern of The first problem encountered, common to most eukaryotic the enzyme from DEAE (Fig. 44).Glycerol gradient sedimenRNA polymeraseisolation procedures, was proteolysis of the tation in high salt eliminated some residual contamination, largest (220-kDa)subunit of polymerase I1 to a truncated 180- but did not separate polymerase I1 from the 700-kDa complex. kDa derivative (Guilfoyle et al., 1984). The method we devel- However, a rapid separation of polymerase I1 from the comoped to minimize proteolysis is simple and appears to be of plex was accomplished, onsmall samples, by agarose electrogeneral applicability. A second problem was the reproducible phoresis, but notwithout a significant loss of activity. Prelimassociation between RNA polymeraseI1 and a 700-kDa com- inary evidence suggests that the complex may be separated plex of 12 polypeptides, which persisted through several chromatographic procedures. We decided to analyze the complex Portions of this paper (including “Materials and Methods” and in some detail, after separating it from the enzyme by prepar- Figs. 1-5) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass.

* This work was supported by National Institutes of Health Grant Full size photocopies are available from the Journal of Biological GM28331 (to S. R. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed.

Chemistly, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-1173, cite the authors, and include a check or money order for $4.80 per set of photocopies. Full size photocopies are also is available from included in the microfilm edition of the Journal that Waverly Press.

16169

16170

Purification of Three Nuclear

RNA Polymerases from

N. crassa

TABLE I Purification of R N A polymerases I, 11, and 111from 130 g (wet weight)of Neurospora mycelia Enzyme fraction

Crude extract Polyimine P eluate Ammonium sulfate precipitate Ion filtration pool Heparin pools DEAE pools Glycerol gradients (the 3 peak fractions)

Enzymes present

Volume ml

1-111 1-111 1-111 1-111 I, I1 111 I I1 I11 I I1 I11

1270 735 60 285 150 119 27 65 39 2.5 2.5 2.5

Activity

Specific activity

m&!

units

unitslmg

6500 3410 1160 400 24.3 12.5 1.0 3.9 0.94 0.28 1.52b 0.21

1590 1650 ND" 1448 830 314 41 648 225 20 360 123

0.245 0.484 ND 3.62 34.2 25.1 41 166 239 70 237 586

Protein

ND, not determined.

* A t this stage, about 40% of the protein

is polymerase II,60% is 700 kDa complex.

from polymerases I and I1 without loss of activity by inserting chromatography on SephacrylS-200 between the heparin and DEAE chromatography steps. Overall Purification and Recouery-The procedure described yielded about 300pgof polymerase I, 600pgof polymerase I1 (after removal of the multi-subunit complex), and 300 pg ofpolymerase 111, starting from 130 g (wet weight) of mycelia (Table I). A direct assessment of the individual enzyme activities in the crude extract was not attempted, since Neurospora polymerase 11, like the homologous enzyme from the mushroom Agaricus bisporus (Vaisius and Horgen, 1979), is resistant tohigh concentrations of a-amanitin, commonly used to differentiate polymerase I1 activity from that of the othertwo. (We foundthat Neurospora polymerase I1 is inhibited only by 35% in presence of 40 pg/ml a-amanitin, whereas Drosophila polymerase I1 is completely inactivated by 1 pg/ml a-amanitin.) The relative activity of each enzyme in the crude extract, then,was roughly estimated on thebasis of the yields from the heparin and DEAE columns (Table I) and from other preparations (not shown). Approximately 10% of the total was assumed to represent polymerase I, 65% polymerase 11, and 25% polymerase 111. Based on the above estimates, at the glycerol gradient stage polymerase I was purified 3,000 x witha12% yield, polymerase I1 (before agarose electrophoresis) was purified 1,500 X with a 35% yield, and polymerase I11 was purified 10,000 x with a 30% yield. Peptide Composition and Molecular Weights-The peptide patterns of the threepurified RNA polymerases from Neurospora and of the complex copurifying with polymerase I1 are presented in Fig. 6. Both thenonproteolyzed and theproteolyzed forms of polymerase I1 are shown. Polymerase I is composed of 10 major subunits, polymerase I1 of9, and polymerase I11 of 15, ranging in molecular mass between 10 and 220 kDa. The 700-kDa complex is composed of 12 polypeptides between 24 and 135 kDa. On Coomassie staining, only polymerase I shows some background contamination (Fig. 6,lane a), mostly by a complex analogous to that found in cruder polymerase I1 preparations. The overall subunit composition of the threepurified Neurospora RNA polymerases, after theseparation of the 700-kDa complex from I and 11, is similar to thatfound in theenzymes from yeast (Hager et al., 1977; Valenzuela et al., 1976) and in Acanthamoeba (D'Alessio et al., 1979). The subunits with the same electrophoretic mobility in the threeenzymes are probably identical (Armaleo and Gross, 1985). The bands appearing in all lanes with varying intensity

700 KD

II

I " r

200 rr 140 r).

.

,..

05

COMPLEX

f l ==

-46

i

10

a

j

'

b

c

FIG. 6. Polypeptide composition of the purified polymerases and 700-kDa complex. SDS-polyacrylamide gel electrophoresis of glycerol gradient-purified polymerases I and I11 (lanes a and d, respectively) and of agarose gel-purified polymerase I1 and complex (lane b, proteolyzed polymerase II; lane c, mostly intact polymerase 11; lane e, 700-kDa complex). Each sample (25-50 pg of protein) was denatured by boiling for 2 min in 70 pl of SDS sample buffer (Laemmli, 1970) and applied into the wells (1.5 X 4 X 25 mm) of a 15 X 14 X 0.15 cm, SDS-polyacrylamideslab gel composed of a 2-cm wide stacking layer and an 11.5-cm wide 12% polyacrylamide resolving gel, modified as described under "Materials and Methods." Electrophoresis was performed a t a constant current of 20 mA until the bromphenol blue had reached the bottom. The molecular masses of the subunits (numbers indicate kDa) were estimated by comparisons with known standards. The arrows in lane a point to subunits of the 700-kDa complexcontaminating the polymerase I preparation.

around 62 kDa and diffusely around 56 kDa are artifacts resulting from the use of 2-mercaptoethanol in the gel sample buffer (Tasheva and Dessev, 1983).The relative subunit staining intensities suggest that most polypeptides are represented once per molecule, as has been found in other cases (Stunnenberg et al., 1979; Spindler et al., 1978; Valenzuela et al.,

Purification of Three Nuclear RNA Polymerases from N . crassa 1976); the 44-kDa subunits in polymerases I and 111 appear to be represented twice, whereas the 65-kDa subunit in I, the 46- and 15-kDa subunits in11, and the 75-kDa subunit in 111 appear in substoichiometric amounts. The “native” molecular masses estimated by adding together the numbers listedin Fig. 6 are 556 kDa for polymerase I, 525 kDa for polymerase 11, 757 kDa for polymerase 111, and 735 kDa for the complex. The estimates for the threeNeurospora enzymes are ingood agreement with those obtained for other eukaryotic RNA polymerases (Lewis and Burgess, 1982). The approximate molecular masses of polymerase 11 and of the complex were determined also by nondenaturing electrophoresis through acrylamide gels of varying porosities (Hedrick and Smith,1968);the values obtained were 480 kDa for polymerase 11 and 730 kDa for the complex. Proteolysis and Evolutionary Conservation of the 220-kDa Subunit of Polymerase 11-The close relatedness between the 220- and 180-kDa subunits of Neurospora polymerase I1 was confirmed by the identity of their peptide maps, obtained by the method of Cleveland et al. (1977) (not shown). This finding, together with the inverse relationship between the amounts of the 220-kDa versus the 180-kDa subunits seen on SDS’ gels of various preparations (see polymerase 11 lanes in Fig. 6) and the effectiveness of early proteolysis prevention, suggests that the180-kDa peptide is generated through cleavage of the 220-kDa subunit, mostly at the very beginning of extraction. The Neurospora enzyme thus conforms to the common pattern of RNA polymerase I1 proteolysis in eukaryotes (Guilfoyle et al., 1984). The-structural propertiesof the 220- and 180-kDa polypeptides were also tested immunologically.Heterologous antibodieswere used, since sera raised against Neurospora RNA polymerases or their subunits are not yet available. After denaturation and transfer onto nitrocellulose (Towbin et al., 1979), the Neurospora subunits were allowed to react with antisera raised specifically against the 220-kDa subunit of RNA polymerase I1 from Drosophila melanogaster or from wheat germ. Whereastheseantisera have been shown to recognize both the 220- and the 180-kDa subunits from the homologous enzymes (Weeks et al., 1982); in our test they reacted only with the 220 kDa and not with the 180-kDa subunit from Neurospora polymerase I1 (not shown). The correlation between the proteolytic loss of a 40-kDa segment and the loss of antigenicity towards heterologous antibodies suggests that theprotease-sensitive regions contribute to phylogenetically highly conserved antigenic determinants on the 220-kDa subunit of type I1 RNA polymerases. A similar conclusion has been reached by Carroll and Stollar (1983). The function of the region sensitive to proteolysis remains poorly defined. Preliminary evidence suggests that itmay be: 1)specifically phosphorylated (Dahmus, 1981); 2) involved in DNA binding (Carroll and Stollar, 1983);and 3) involved in directing specific transcription, in vitro,from the adenovirus2 major late promoter, as well as the conalbumin and the ovalbumin promoters (Dahmus and Kedinger, 1983). The rapid proteolytic generation of an essentially unique 180-kDa species resistant to further proteolysis (Guilfoyle et al., 1984) and the relative insensitivity of most of the basic catalytic propertiesof polymerase I1 to theloss of the cleaved regions (Dezelee et al., 1976) suggest that in the native enzyme the 40-kDa terminal region of the 220-kDa subunit is at the The abbreviations used are: SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; MeZSO,dimethyl sulfoxide; BSA, bovine serum albumin; TCA, trichloroacetic acid; PAGE, polyacrylamide gel electrophoresis. A. Greenleaf, personal communication.

16171

surface of the molecule and is, to some extent, independent from the structure of the rest of the polymerase. It has been argued that intron-exon junctions tend tocorrespond to sections of the polypeptide chain located on the surface of the native protein (Craik et al., 1982). Three introns have been found within the coding sequence of the gene for the 220-kDa subunit of Drosophila polymerase I1 (Biggs et al., 1985). The position of one of them limits a section at the 3’-end of the gene which can accommodate a carboxyl-terminal polypeptide segment of 40-50 kDa. It remains to be demonstrated whether this segment does in fact correspond to the proteolyzed domain. Heterogeneities in Polymerase I I Preparations-Besides the “220 kDa/l80 kDa” dimorphism and the association with varying amounts of 700-kDacomplex, two other types of structural heterogeneity were observed in preparations of polymerase 11. One yielded a well-defined polymerase doublet on nondenaturing polyacrylamide gels (see Fig. 4A in Armaleo and Gross, 1985). SDS gel analysis revealed that the fastermigrating form was missing the 15-kDa subunit, whereas the slower species contained the 15-kDa subunit (not shown). No other difference was apparent. The polymerase doublet seen here on native gels, unlike the electrophoretic dimorphism commonly found in typeI1 polymerases (Dezelee et al., 1976), did not appear to be related to proteolysis of the 220-kDa subunit. Polymorphisms related to the variable presence of particular “subunits” have been reported for polymerase I (Matsui et al., 1976; Huet et al., 1975). The heterogeneity described could account for the substoichiometric amounts of the 15-kDa polypeptide in purified Neurospora polymerase I1 preparations (Fig. 6, lanes b and c). The other kind of heterogeneity closely resembles one of the subunit changes described for polymerase I1 from Saccharomyces cerevisiue, which is found variably associated with a 16.5- and a 32-kDa protein. These peptides, on native gels, migrate ahead of the enzyme as a single high mobility species (Dezelee et al., 1976;Ruet et al., 1980). Native gels of partially purified Neurospora polymerase I1 preparations revealed, in addition to the enzyme and the 700-kDa complex, a third major high mobility species. Upon SDS gel electrophoresis in the second dimension, this species dissociated into a 35-kDa protein and a closely spaced 12- and 12.5-kDa doublet (data not shown). Acknowledgments-Wewouldparticularly like to thank Arno Greenleaf, Doug Coulter, Joe Biggs, Brett Tyler, and Geoffrey Kidd for the antibodies and for many useful discussions and Gerda Vergara for excellent technical assistance. Note Added in Proof-The recent sequencing of the gene for the largest subunit of yeast polymerase I1 (Allison et al., 1985) has established boththe evolutionary conservation and the carboxylterminal location of the proteolyzed domain of the 220-kDa polypeptide. REFERENCES Allison, L. A., Moyle, M., Shales, M., and Ingles, J. (1985) CeU 4 2 , 599-610 Armaleo, D., and Gross, S. R. (1985) J.Biol. Chem. 260,611-621 Biggs J., Searles L. L., and Greenleaf, A. L. (1985) Cell 42,16174-16180 Bradf’ord, M. (19:s) Anal. Biochem. 72,248-254 Carroll, S. B., and Stbllar, B. D. (1983) J. Mol. Biol. 170,777-790 Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977) J.Biol. Chem. 252,1102-1106 Craik, C. S., Sprang, S., Fletterick, R., and Rutter, W. J. (1982) Nature 299, 1“” N L l F(7 &”

Dahmus, M. E. (1981) J. Biol. Chem. 256,3332-3339 Dahmus, M. E., and Kedinger C.(1983) J. Biol. Chem. 258,2303-2307 DAlessio, J. M., Perna, P. J.: and Paule, M. R. (1979) J. Biol. Chem. 254, 11282-11287 Davis, R. H., and de Serres, F. J. (1970) Methods Enzymol. 17, 79-143 Dezelee, S., Wyers, F., Sentenac, A,, and Fromageot,P. (1976) Eur. J.Biochem. 65,543-552 Guilfoyle, T. J., Hagen, G., and Malcom, S. (1984) J. BioL Chem. 259, 649653

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Purification of Three Nuclear RNA Polymerases from N. crassa

Ha er, G. L., Holland, M. J., and Rutter, W. J. (1977) Biochemistry 1 6 , l - S H e Lck J. L., and Smith,A. J. (1968) Arch. Biochem. Biophys. 126,155-164 Huet J.'Buhler, J-M. Sentenac, A., andFromageot, P. (1975) Proc. Natl. Acad. Sc: d. S. A. 72,30343038 Jendrisak, J. J., and Bur ess, R. R. (1975) Biochemistry 14,4639-4645 Kaehn, K., Morr, M., and Kula, M-R. (1979) Hoppe-Seyler's Z.Physzol. Chem. 360, 791-794 Kirkegaard, L. H., Johnson, T. J. A., and Bock, M. (1972) Anal. Biochem. 50, 122-138 Laemmli, U. K. (1970) Nature 227,680-685 Lewis, M. K., and Burgess, R. R. (1982) in The Enzymes (Boyer, P. D., ed), 109-153, Academic Press, New York M:t%:$% , shi, T., and Muramatsu, M. (1976) Eur. J. Biochem. 71, 351360 Mendez. E. (1982)Anal. Biochem. 126.403-408 Macart 'M., and Gerbaut, L. (1982) Cliiz. Chin. Acta 122,93-101 Roeder,' R. G. (1976) in RNA Polymerase (Losick and Chamberlin, eds), pp. 285-329 Cold Spring HarborLaboratory, Cold Sprin Harbor, NY Ruet. A.. Sentenac. A.. Fromaseot. P.. Winsor, B.. an8Lacroute. F. (1980) J. Bwl. Chem. 255; 6450-6455"

Smith, S. S., and Braun, R. (1978) EUI:J. Biochem. 82,309-320 Spindler, S. R., Duester, G. L., D'Alessio, J. M., and Paule, M. R. (1978) J. BioL Chem 253,4669-4675 Studier, F. W. (1973) J. Mol. Biol. 79,237-248 Stunnenberg, H. G., Wennekes, L. M. J., and Van Den Broek, H. W. J. (1979) Eur. J. Biochem. 98,107-119 Tasheva, B., and Dessev, G. (1983) A d . Biochem. 129,98-102 Tellez de Inon, M. T., Leoni, P. D., and Torres, H. N. (1974) FEBS Lett. 39, 91-95 Timberlake, W. E., and Turian,G. (1974) Erperientiu 30,1236-1238 Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci U. S. A. 76,4350-4354 Tyler, B. M., Geever, R. F., Case, M. E., and Giles, N. H. (1984) Cell 36,493502 Vaisius, A. C., and Horgen, P. A. (1979) Biochemistry 18, 795-803 Valenzuela,P., Hager, G. L., Weinherg, F., and Rutter,W. J. (1976) Proc. Natl. Acad. Sci. U. S. A. 73,1024-1028 Weeks, J. R., Coulter, D. E., and Greenleaf, A. L. (1982) J. Biol. Chem. 257, 5884-5891

ENZ*ldE RJRIFICATION P M I PREINCU8AlION

1

CRUDEEXTRACT I-IS'I POLYETHYLENEIMINEPRECIPlTATlON

i t .ION-FILTRATION CHROMATOGRAPHY

AMMONIUM SULFAIE PREClflTAllON

t

HEPARIN-AGAROSE CHROMATOGRAPHY

/ 11.111

\11111 1

/

DEI€-SEPHADEX CHROMATOGRAPHY

DEAE-SEPHADEX CHROMATOGRAPHY

t

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t

i i AGAROSE GEL

11111

I

\

GLYCEROL

\ (11

1

GRADIENT SEDIMENTATION

1111

t

ELE~TROPHO~ESIS

/

1111 Fig. 1

\

\

1700 KDg COMPLEX)

8

7 .

Purification of Three Nuclear R N A Polymerases from N. crassa

lot

IO 9

K

GELSLICES

700 K O COMPLEX

3

> c

0.2 0

20

30 40 50 60 70 FRACTION NUMBER

80

POLYMERASE I I

16173