Communicated by Kenneth B. Raper, January 7, 1974. ABSTRACT. Two RNA .... Present address: Noland Zoology Building,University of. Wisconsin, Madison ...
Proc. Nat. Acad. Sci. USA
Vol. 71, No. 4, pp. 1174-1177, April 1974
Purification and Properties of Two RNA Polymerases from Physarum polycephalum (slime mold/polypeptide subunits)
ANN BAKER BURGESS* AND RICHARD R. BURGESS McArdle Laboratory for Cancer Research, University of Wisconsin Medical Center, Madison, Wisc. 53706 Communicated by Kenneth B. Raper, January 7, 1974
Two RNA polymerases have been purified ABSTRACT from the slime mold Physarum polycephalum, one sensitive and one resistant to a-amanitin. Both enzymes are more active with denatured DNA than native DNA as a template and prefer Mn++ rather than Mg++ as a divalent cation. The a-amanitin-sensitive enzyme shows maximum activity at 0.15 M KCI, whereas the resistant enzyme is most active at very low ionic strength. Analysis of the resistant enzyme on polyacrylamide gels containing sodium dodecyl sulfate shows two subunits present in a 1:1 ratio with molecular weights of 205,000 and 125,000.
By now, multiple species of RNA polymerase have been isolated from a number of eukaryotes (1-5), including simple ones such as yeast and slime molds. The enzymes from a particular organism or tissue generally differ in the salt concentration at which they elute from DEAE-cellulose columns, their sensitivity to the mushroom toxin a-amanitin, and the ionic strength and divalent cation required for maximum activity. RNA polymerase I, which is not inhibited by aamanitin, is localized in the nucleolus and is generally believed to be responsible for ribosomal RNA synthesis. Polymerase II, on the other hand, is completely inhibited by small amounts of a-amanitin and is localized in the nucleoplasm. Some organisms have a third RNA polymerase, which is also localized in nucleoplasm but insensitive to a-amanitin (6). In this report we will describe the purification and some of the properties of two RNA polymerases from the plasmodial stage of the slime mold Physarum polycephalum.
Enzyme Assay. The standard RNA polymerase assay con-
tained, in 0.2 ml, 0.05 M Tris, pH 7.9, 0.075 mg/ml of heatdenatured calf-thymus DNA (Worthington), 0.6 mM each ATP, CTP, and GTP (P-L Biochemicals), 2 ,Ci of [8H]UTP, 27 Ci/mmol (New England Nuclear Corp.), 40 sg/ml of pyruvate kinase (Calbiochem), 2.5 mM phosphoenol pyruvate, 1 mM dithiothreitol, 1 mM MnCl2, and 0.1 M KCl. The UTP concentration in the assay was only 0.4 uM in order to increase the sensitivity of the assay by maintaining a high specific activity. The reaction mixture was incubated for 30 min at 30°, chilled, and precipitated with 5% trichloroacetic acid containing 5 mM sodium pyrophosphate. The precipitate was collected on a glass-fiber filter, washed with 2% trichloroacetic acid, dried, and counted in toluene-2,5-diphenyloxazole. One unit of activity is the amount of enzyme that will catalyze the incorporation of 1 pmol of UMP into RNA in 30 min at 30°. It is equivalent to 104 cpm incorporated. Protein concentration was determined by the method of Lowry et al. (9) or, for more purified fractions, by measurement of the absorbance at 280 nm. Specific activity is in units/mg of protein. The a-amanitin used in some assays was the gift of Professor Wieland.
02
METHODS
x
Organism. Physarum polycephalum, strain M3c, was obtained from Dr. Joyce Mohberg and grown either as microplasmodia shaken in liquid medium or as macroplasmodia on filter paper in rocker pans (7). In either case, it is necessary to harvest cells in early logarithmic phase in order to obtain maximum RNA polymerase activity. Nuclei were prepared according to Procedure B of Mohberg and Rusch (8), with MgCl2 in the homogenizing medium and omission of the final wash. They were resuspended in an equal volume of 50%O glycerol in 0.05 M Tris (pH 7.9)-5 mM MgCl2-0.1 mM EDTA-1 mM dithiothreitol and stored at -80° until use. Nucleoli were prepared as described in this same reference. All operations were performed at 0-4'.
00
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n a.
5
10
15
20
FRACTION NUMBER
FIG. 1. Glycerol gradient profile. Dialyzed crude extract (1.6 ml) (7 mg of protein per ml) was layered on a 15-30% glycerol gradient and centrifuged at 90,000 X g for 36 hr. Fractions (1.7 ml) were collected, and 0.05-ml aliquots were assayed for RNA polymerase activity. Fractions 10-12 were pooled with similar fractions from three other gradients. The arrow shows the position of the ,-galactosidase marker. The bottom of the gradient is at the left. (O--*) RNA polymerase activity; (--- ) A280.
Abbreviation: TGED buffer, 0.05 M Tris (pH 7.9)-25% glycerol0.1 mM EDTA-1 mM dithiothreitol. * Present address: Noland Zoology Building, University of Wisconsin, Madison, Wisc. 53706. 1174
Proc. Nat. Acad. Sci. USA 71
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Physarum RNA Polymerases
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TABLE 1. Purification of RNA polymerases I and II from Physarum polycephalum
to
10 x
a. Q a
Total activity units
2
Fraction
w
0
0 cL)
C-0.10
2
30 25 20 I5 FRACTION NUMBER FIG. 2. DEAE-cellulose column profile. Protein (4.8 mg) from the glycerol gradients was applied to a 1 X 8-cm column of DEAE-cellulose and eluted with a 70-ml gradient as described in the text. Fractions (1.8-ml) were collected and 0.05-ml aliquots were assayed for activity. Fractions 12-17 were pooled. (@--*) 5
10
RNA polymerase activity; (0- - -0) A2uo; ( tration.
) KCl
concen-
Solubilization of RNA Polymerase. The procedure of Roeder and Rutter (6) was used to solubilize RNA polymerase, remove chromatin, and concentrate the crude extract, except that nuclei were sonicated in 10-ml aliquots in the buffer in which they were frozen, with the addition of (NH4)2S04 to 0.3 M. In some cases phenylmethanesulfonyl fluoride was added to inactivate proteolytic enzymes (10), but no difference in stability was detected when this was done. More recently we have sonicated nuclei in 0.5-1.0 M NH4Cl rather than 0.3 M (NH4)2S04, resulting in an increased yield of RNA
1470 Crude extract Pooled glycerol gradient 880 peak Pooled DEAE-cellulose 360 column peak Phosphocellulose column Peak 1 (RNA 64 polymerase II) Peak 2 (RNA 310 polymerase I)
Specific activity
A280/
A2*0
Yield (%)
34
0.78
100
260
0.88
60
380
1.5
24
*20,000
4
*35,000
21
* The specific activities at the final stage of purification are only rough estimates because the amount of protein present was determined as explained in the legend to Fig. 6.
KCl, centrifuged to remove any proteins that precipitated, and layered over 15-30% linear glycerol gradients in this same buffer. The gradients were centrifuged at 4° for 36 hr at 26,000 rpm in the Spinco SW 27 rotor and collected through a recording spectrophotometer. Column Chromatography. DEAE-cellulose and phosphocellulose columns were prepared and equilibrated as described (11), except that the buffer used was 0.05 M Tris (pH 7.9)25% glycerol-0.1 mM EDTA-1 mM dithiothreitol (TGED) plus KCl as noted.
polymerase. Glycerol Gradient Centrifugation. The crude extract was dialyzed for several hours against 8% glycerol in 0.05 M Tris (pH 7.9)-0.1 mM EDTA-1 mM dithiothreitol-0.2 M
6
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A
B
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2
4
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22 I aC.) w
cr a. 0
0
Z C.- 4
24
0.I
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15
10
20
25
30
FRACTION NUMBER
FIG. 3. Phosphocellulose column profile. Protein (0.9 mg) from the DEAE-cellulose column was applied to a 1 X 4-cm phosphocellulose column at a flow rate of 5 ml/hr and eluted with a 40-ml gradient as described in the text. Fractions (1.5-ml) were collected, and 0.05-ml aliquots were assayed for activity in the absence or presence of 2.5 ug/ml of a-amanitin. (@--*) RNA polymerase activity; (A--A) a-amanitin-resistant RNA polymerase activity; (O- 0) A280; (--) KCl concentration. - -
5
10
CATION
-1I10
(mM)
FIG. 4. Effect of salt and divalent cations on the activity of separated RNA polymerases. Aliquots of phosphocellulose column fractions that had been concentrated by ultrafiltration were incubated under standard assay conditions, except that either KCl concentration (A and B) or MnCl2 or MgCI2 concentrations (C and D) were varied. (A and C) RNA polymerase from the first phosphocellulose column peak (RNA polymerase II). (B and D) RNA polymerase from the second phosphocellulose column peak (RNA polymerase I).
1176
i.5
10
Proc. Nat. Acad. Sci. USA 71
Biochemistry: Burgess and Burgess A
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-
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DNA CONCENTRATION, ug/ ml FIG. 5. Effect of DNA on the activity of separated RNA polymerases. Enzyme fractions similar to those described in Fig. 4 were incubated under standard assay conditions with various amounts of native or denatured calf-thymus DNA. (I@-*) RNA polymerase activity in the presence of denatured DNA; (0- -0) RNA polymerase activity in the presence of native DNA. (A) RNA polymerase II; (B) RNA polymerase I. -
Polyacrylamide Gel Electrophoresis. Electrophoresis was performed on 8.75% polyacrylamide gels containing sodium dodecyl sulfate according to the method of Laemmli (12). Molecular weights of RNA polymerase subunits were determined by comparing their mobilities with those of a series of marker proteins of known molecular weight run on a parallel gel. RESULTS AND DISCUSSION
Purification of Two RNA Polymerases. Packed nuclei (41 ml) from 64 macroplasmodia were pooled and a crude extract was prepared. This material was centrifuged on four glycerol gradients, and the peak fractions of RNA polymerase activity were pooled. As can be seen from Fig. 1, one peak of RNA polymerase activity was obtained, coinciding with the position of j3-galactosidase at 16 S centrifuged on a parallel gradient. We routinely recover 100% of the RNA polymerase activity put onto the gradients, although only about 60% is present in the tubes that are pooled. The purification is summarized in Table 1. The pooled glycerol gradient peak fraction was diluted with 25% glycerol until the conductivity was less than that of
(1974)
TGED buffer containing 0.08 M KCl and applied to a DEAEcellulose column that had been equilibrated with TGED buffer containing 0.08 M KCl. The column was washed with this same buffer, and then a KCl gradient from 0.08 to 0.6 M was applied. One peak of RNA polymerase activity was obtained (Fig. 2), which eluted at 0.16 M KCl and was partially sensitive to a-amanitin. We have not been able to separate the a-amanitin-sensitive and -resistant activities on DEAEcellulose or on Sephadex using a variety of salts and much shallower gradients than that shown in Fig. 2. This result contrasts with Lost other organisms where DEAE-cellulose chromatography has been the method used to separate the two activities. When an extract from isolated nucleoli was chromatographed on DEAE-cellulose, a peak of completely a-amanitin-resistant activity eluted at the same salt concentration as the mixture of enzymes from nuclei. Although the DEAE-cellulose column results in only a small increase in specific activity (Table 1), it is an important step because it removes nucleic acid. If this is not done, the enzymes fail to bind to phosphocellulose in the next step of the purification. The material from the pooled DEAE-cellulose peak was diluted with 25% glycerol until the conductivity was less than that of TGED buffer containing 0.15 M KCl and applied to a phosphocellulose column that had been equilibrated in TGED buffer containing 0.15 M KCl. The column was washed, and a gradient from 0.15 to 0.9 M KCI was applied. As can be seen from Fig. 3, two peaks of RNA polymerase activity were obtained. The material of the first peak, which eluted at 0.28 M KCl, was sensitive to a-amanitin and will be referred to as RNA polymerase II, since that designation has been used in other systems for the a-amanitin-sensitive enzyme. The material in the second peak, which eluted at 0.54 M KCl, was resistant to a-amanitin and will be referred to as RNA polymerase I. The relative amounts of these two enzymes vary from one preparation to another, which may reflect the growth state of the cell (13) or the efficiency of extraction of the enzymes during sonication of nuclei in high salt. A curious finding, which we cannot explain, has been that we consistently recover more a-amanitin-resistant activity than we put on the phosphocellulose column and less a-amanitin-sensitive activity. n
E D
*1|11
Co IA
FIG. 6. Polyacrylamide gels containing sodium dodecyl sulfate of Physarum RNA polymerase at various stages of purification. (A) Protein (68 Mug) from a crude nuclear extract. (B) Protein (24 jig) from glycerol gradient peak. (C) Protein (27 fig) from DEAE-cellulose column peak. (D) Protein from phosphocellulose column peak 1 (RNA polymerase II). (E) Protein from phosphocellulose column peak 2 (RNA polymerase I). (F) Molecular weight marker proteins: E. coli RNA polymerase fl' (165,000) and # (155,000) subunits, phosphorylase a (94,000), E. coli sigma factor (87,000), bovine-serum albumin (68,000), pyruvate kinase (57,000), ovalbumin (43,000), E. coli RNA polymerase a subunit (39,000), chymotrypsinogen (25,700). The amounts of protein in D and E are estimated to be 0.5 and 1 jug, respectively, on the basis of comparison of the amount of stain in the bands with that of known amounts of protein.
Proc. Nat. Acad. Sci. USA 71
(1974)
.I FIG. 7. Densitometer tracing of the gel shown in Fig. 6E. The top of the gel (on the left in Fig. 6E) is shown at the left.
Properties of RNA Polymerases I and II. The response of the two enzymes to variations of ionic strength and divalent cation is shown in Fig. 4. RNA polymerase II exhibits its maximum activity at higher salt than does RNA polymerase T, in common with the two enzymes from other sources such as rat liver or sea urchin (1). Both enzymes require a divalent cation for activity and prefer Mn++ over Mg++. Both RNA polymerases prefer denatured DNA as a template when tested with calf-thymus DNA (Fig. 5). Analysis of Physarum RNA polymerase at various stages of purification on polyacrylamide gels is shown in Fig. 6. At the last step in the purification, only RNA polymerase I is pure enough to determine its subunit structure. Two high-molecular-weight subunits are apparent which have molecular weights of 205,000 and 125,000. Scanning the gel with a recording spectrophotometer and measuring the area under the two peaks show that the subunits are present in a 1:1 ratio (Fig. 7). Smaller proteins are evident on the scan at positions corresponding to molecular weights of 60,000 and about 45,000, but they are present in such low amounts that it is impossible to say whether they are subunits or impurities. The highest molecular-weight band on the gel (250,000) has been
Physarum RNA Polymerases
1177
identified as myosin (14) by comparing its position with that of myosin from Physarum nuclei subjected to electrophoresis on a similar gel (W. LeStourgeon, personal communication). Hildebrandt and Sauer (5) recently reported the isolation of a-amanitin-sensitive and -resistant RNA polymerases from Physarum using DEAE-cellulose chromatography. We cannot explain the difference in chromatographic properties of their enzymes and ours, but the other properties of the enzymes appear to be similar. We thank Ms. Debbie O'Brien for excellent technical assistance and Dr. Joyce Mohberg for much helpful advide. This work was supported by Grant CA-07175 from the National Cancer Institute. 1. Roeder, R. G. & Rutter, W. J. (1969) Nature 224, 234-237. 2. Blatti, S. P., Ingles, C. J., Lindell, T. J., Morris, P. W., Weaver, R. F., Weinberg, F. & Rutter, W. J. (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 649-657. 3. Chambon, P., Gissinger, F., Mandel, J. L., Kedinger, C., Gniazdowski, M. & Meihlac, M. (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 693-707. 4. Pong, S. S. & Loomis, W. F. (1973) J. Biol. Chem. 248, 3933-3939. 5. Hildebrandt, A. & Sauer, H. W. (1973) FEBS Lett. 35, 4144. 6. Roeder, R. G. & Rutter, W. J. (1970) Proc. Nat. Acad. Sci. USA 65, 675-681. 7. Mohberg, J. & Rusch, H. P. (1969) J. Bacteriol. 97, 14111418. 8. Mohberg, J. & Rusch, H. P. (1971) Exp. Cell Res. 66, 305316. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 10. Pringle, J. R. (1970) Biochem. Biophys. Res. Commun. 39, 46-52. 11. Burgess, R. R. (1969) J. Biol. Chem. 244, 6160-6167. 12. Laemmli, U. K. (1970) Nature 227, 680-685. 13. Grant, W. D. (1972) Eur. J. Biochem. 29, 94-98. 14. Nachmias, V. T. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 607-612.
