Proc. Nat. Acad. Sci. USA Vol. 73, No. 4, pp. 1029-10&3, April 1976 Biochemistry Transcription in yeast: a-Amanitin sensitivity and other properties which distinguish between RNA polymerases I and III* (chromatography on DEAE-ion-exchangers/salt-activation profiles/RNA nucleotidyltransferase) LOREN D. SCHULTZt AND BENJAMIN D. HALL* t Department of Biochemistry and t'Department of Genetics, University of Washington, Seattle, Wash. 98195 Communicated by Herschel L. Roman, December 29, 1975 ABSTRACT Three peaks of DNA-dependent RNA poly- umns can be interpreted either as a failure of DEAE-cellu- merase (RNA nucleotidyltransferase) activity are resolved by lose to resolve two different enzymes or, alternatively, as the chromatography of a sonicated yeast cell extract on DEAE- Sephadex. The enzymes, which are named RNA polymerases tendency of a single enzyme, polymerase A, to chromato- I, II, and III in order of elution, show similar catalytic prop- graph as two peaks on DEAE-Sephadex as a result of some erties to the vertebrate class I, class II, and class III RNA po- trivial alteration that occurs during the chromatography. lymerases, respectively. Yeast RNA polymerase III is readily For vertebrate RNA polymerases, the first of these interpre- distinguished from yeast polymerase I by its biphasic ammo- tations is the correct one. Sklar, Schwartz, and Roeder (10) nium sulfate activation profile with native DNA templates, have shown, for mouse plasmacytoma RNA polymerases, greater enzymatic activity with poly[d(I-C) than with native that each of the three classes of RNA salmon sperm DNA, and distinctive chromatographic elution polymerase eluted positions from DEAE-cellulose (0.12 M ammonium sulfate) from DEAE-Sephadex has a distinctive pattern of protein compared with DEAE-Sephadex (0.32 M ammonium sulfate). subunits. The failure of two of these three enzymes to be re- The three yeast RNA polymerases also show significant solved on DEAE-cellulose has been demonstrated directly differences in a-amanitin inhibition. RNA polymerase II is (11-14). the most sensitive (50% inhibition at 1.0 gg of a-amanitin per To determine whether the three-peak pattern of yeast ml). Contrary to the results for vertebrate systems, yeast poly- RNA polymerases also is truly indicative of three distinct en- merase I can be completely inhibited by a-amanitin at high concentrations (50% inhibition at 600 gg/ml) while yeast zymes, we have done experiments on the rechromatography RNA polymerase III begins to show significant inhibition of DEAE-Sephadex peaks on DEAE-cellulose and vice versa. only at concentrations exceeding 1 mg/ml. Therefore, yeast The results of these experiments, which we report here, indi- RNA polymerases I and III show a pattern of a-amanitin sen- cate that there are three yeast enzymes, each having quite sitivity that is the reverse of that seen for the analogous ver- distinctive catalytic and chromatographic properties. tebrate RNA polymerases. In their recent structural studies of homogeneous yeast Multiple forms of eukaryotic RNA polymerase (RNA nu- RNA polymerases I, II, and III, Valenzuela, Hager, cleotidyltransferase), which were first found to exist in sea Weinberg, and Rutter (15) have demonstrated that yeast urchin nuclei (1, 2), have since been found in many differ- RNA polymerase III has a pattern of high-molecular-weight ent species of animals, plants, and fungi (3). The number of protein subunits similar to that observed for vertebrate RNA distinct classes of eukaryotic nuclear RNA polymerase re- polymerase III and quite different from those found for ported has most often been three in studies where DEAE- yeast RNA polymerases I and II (15-18). The protein sub- Sephadex was used for enzyme fractionation and two in unit data, together with the catalytic and chromatographic those studies using DEAE-cellulose (reviewed in ref. 4). In studies we report here, indicate that there are three distinct their investigation of the RNA polymerases of KB (human) yeast RNA polymerases. Each of these three enzymes resem- cells, Sergeant and Krsmanovic (5) chromatographed paral- bles in most of its properties that vertebrate RNA polymer- lel samples of the same extract on DEAE-Sephadex and ase which corresponds to it in DEAE-Sephadex elution be- DEAE-cellulose columns. Three peaks of enzyme activity havior. However, the pattern of a-amanitin resistance of the eluted from the DEAE-Sephadex column and two from three yeast RNA polymerases does not parallel that of the DEAE-cellulose, with the second peak being highly sensitive three vertebrate enzymes. Because of this discordance, the to a-amanitin in both cases. An entirely analogous situation polymerase A, B, C system of nomenclature (4) cannot be exists for the yeast RNA polymerases extracted from nuclei meaningfully applied to yeast RNA polymerases. or from whole cells. Sentenac and coworkers have observed two peaks of yeast RNA polymerase on DEAE-cellulose, the MATERIALS AND METHODS first resistant and the second sensitive to a-amanitin (6), Biochemicals. DEAE-Sephadex (A-25) was obtained while results from this and other laboratories (7-9) have from Pharmacia and DEAE-cellulose (DE-52) from What- shown that DEAE-Sephadex resolves three major peaks of man. Unlabeled ribonucleoside triphosphates were obtained yeast RNA polymerase activity, of which only the second is from P-L Biochemicals. [3H]UTP and [3H]GTP (10-25 Ci/ sensitive to 20 ;tg/ml of a-amanitin. For both the mamma- mmol) were purchased from New England Nuclear. En- lian and yeast RNA polymerases, the differing number of zyme grade ammonium sulfate and Tris-base were obtained components observed on the two types of ion-exchange col- from Schwarz/Mann. a-Amanitin was purchased from both Henley and Co., New York, and Calbiochem. Type V calf thymus DNA was purchased from Sigma, salmon sperm Abbreviation: TGED buffer, 50 mM Tris-HCI (pH 7.9), 25% (vol/ vol) glycerol, 0.5 mM EDTA, 1.0 mM dithiothreitol. DNA from Worthington, poly[d(I-C)] (s2o,0 = 9.0 S) from * These findings were presented at the Cold Spring Harbor Labora- P-L Biochemicals, and poly[d(A-T)] (s20w = 15 S) from tory Meeting on RNA Polymerases (August 19-24, 1975; Cold Miles Laboratories. Dithiothreitol and phenylmethylsul- Spring Harbor, New York). fonylfluoride were obtained from Sigma. 1029 Downloaded by guest on September 29, 2021 1030 Biochemistry: Schultz and Hall Proc. Nat. Acad. Sci. USA 73 (1976) Solutions. Extraction buffer was 0.2 M Tris.Cl (pH 7.9, 10 ~~~~~~A 220), 20% (vol/vol) glycerol, 20 mM MgCl2, 0.8 M 8- (NH4)2SO4, 1.0 mM EDTA, and 1.0 mM dithiothreitol. Im- mediately before use, phenylmethylsulfonylfluoride (34 mM 6- 0.6 stock solution in absolute ethanol) was added to a final con- 4- 0.4 centration of 3.4 mM. TGED buffer was 50 mM Tris-Cl (pH E 7.9, 220), 25% (vol/vol) glycerol, 0.5 mM EDTA, and 1.0 2- 0.2 mM dithiothreitol. -0 - - v rrn-.--.-- 60 80 100 120 140 160 B Yeast Strains, Media, and Cell Growth. Yeast cells were la grown with aeration at 30'. The Saccharomyces cerevtsiae t- 5 E strain used in most of the experiments (except Fig. 4) was .2 4 0.8 E the haploid strain A364A (19), which was grown in YM-1 0 a per For medium (19) to final density of 4 X 107 cells ml. rI 3 0.6 < the experiment in Fig. 4, cells of S. cerevisiae strain Y55, a x E z 0.4 wild-type diploid (20), were grown in YEP medium (20) to a final density of 4 X 107 cells per ml. After harvesting, cells I 0.2 were washed twice with glass distilled water and stored at _ .j. _ -V.VI-on~~~~~~~~~~~~~~~~~~~~~~~~~~- -70 |1.2 .,,_____C_ Enzyme Solubilization. RNA polymerase was solubilized > 0.8 - lro from 3- to 10-g quantities of yeast cells as described (7), with ._ ._ ' I 4I Io ' I. the following modifications: the homogenization mixture WAI 60 80 loo 120 140 160 180 consisted of 1 part yeast cells, 1 part glass distilled water, 2 Fraction Number parts extraction buffer, and 2 parts 0.45 mm glass beads; and FIG. 1. Resolution of multiple forms of RNA polymerase on prior to chromatography, the crude extract was diluted with DEAE-Sephadex and DEAE-cellulose. (A) RNA polymerase was TGED buffer containing 1.7 mM phenylmethylsulfonylfluo- extracted from 5 g of yeast cells as described in Materials and ride. Methods. The sample was chromatographed on a 2.6 X 12 cm Ion Exchange Chromatography. Columns containing DEAE-Sephadex column at a flow rate of 30 ml/hr. Assays were DEAE-Sephadex or DEAE-cellulose were equilibrated with performed on 45-Ml aliquots of the 5.5-ml fractions, as described in TGED buffer containing 50 mM ammonium sulfate. The Materials and Methods. (B) The RNA polymerase activity from 5.2 g of cells was solubilized as in Materials and Methods except sample was applied to the column, and it was washed with that the extraction buffer was 0.4 M ammonium sulfate. The sam- containing 50 mM am- 1.5 column volumes of TGED buffer ple was chromatographed on a 2.6 X 12 cm column of DEAE-cellu- monium sulfate. Proteins were eluted with a linear gradient lose at a flow rate of 30 ml/hr. At the completion of the gradient, from 0.05 to 0.43 M ammonium sulfate in 12 column vol- the column was washed with TGED buffer containing 1.0 M am- umes of TGED buffer. Fractions containing RNA polymer- monium sulfate. Fractions of 5.5 ml were collected and 50-M4l ali- RNA ase activity were concentrated as described (7).