Proc. Nat. Acad. Sci. USA Vol. 73, No. 4, pp. 1029-10&3, April 1976 Biochemistry in yeast: a-Amanitin sensitivity and other properties which distinguish between RNA I and III* (chromatography on DEAE-ion-exchangers/salt-activation profiles/RNA ) 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 or, alternatively, as the chromatography of a sonicated yeast extract on DEAE- Sephadex. The enzymes, which are named RNA polymerases tendency of a single , 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 . 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). quots were assayed for RNA polymerase activity. (0 *), RNA Polymerase Assay. The RNA polymerase activity in polymerase activity; ( ), ammonium sulfate molarity. The flow- through and wash fractions in (A) and (B) contained no detectable column fractions was measured as in Fig. 1 of ref. 7, except activity (results not shown). (C) RNA polymerase activity across that the incubation time was 20 min. Assays were stopped by the DEAE-cellulose column in (B) was reassayed in the presence spotting 75 jul onto 2.3 cm diameter Whatman 3MM paper and absence of 25 sAg of a-amanitin per ml. Assays were as in part discs, which were then immersed for 10 min in cold 10% (B) except 30 Ml of each fraction were assayed in a final reaction C13CCOOH + 0.12 M sodium pyrophosphate. The filters volume of 100 Ml. (A--A), activity in presence of a-amanitin di- vided by activity in absence of a-amanitin. were washed in batches according to Mans and Novelli (21) and radioactivity was measured by liquid scintillation count- initially chromatographed on DEAE-cellulose (Fig. 1B). ing in Liquifluor-toluene (New England Nuclear Co.). Two peaks of RNA polymerase activity were obtained: peak A eluting at 0.12 M and peak B eluting at 0.20 M ammo- RESULTS nium sulfate. Peak B was identified as containing only RNA polymerase II by its complete sensitivity to low concentra- Differential chromatography of yeast RNA tions (25 gg/ml) of a-amanitin (Fig. 1C). The observation on and DEAE-cellulose polymerases DEAE-Sephadex that no third peak of polymerase activity was eluted from DEAE-Sephadex chromatography of yeast cell extracts DEAE-cellulose and the high recovery of RNA polymerase yields three peaks of RNA polymerase activity (Fig. 1A). activity in the peak A region suggested the possibility that The three RNA polymerase activities, eluting at 0.17 M, polymerase I and III activities might both be present in this 0.245 M, and 0.32 M ammonium sulfate, are designated en- peak. The composition of DEAE-cellulose peak A was exam- zymes I, II, and III, respectively, on the basis of the nomen- ined by rechromatography on DEAE-Sephadex (Fig. 2C). clature proposed for the nuclear RNA polymerases of animal Two peaks of RNA polymerase activity were clearly re- cells (1, 2). As has been shown for animal cell RNA polymer- solved, eluting at the ammonium sulfate concentrations at ases (1, 2, 11, 22), yeast RNA polymerases I and III retain which polymerases I and III normally elute off DEAE-Se- their characteristic chromatographic properties when re- phadex. The ionic strength optima for the polymerase activi- chromatographed on DEAE-Sephadex; yeast RNA polymer- ties in the first and second peaks were similar to those ob- ases I and III continue to elute at 0.17 M and 0.32 ammo- served for polymerases I and III, respectively. These results nium sulfate, respectively (Fig. 2A and B). RNA polymerase indicate that yeast RNA polymerases I and III cochromato- II also rechromatographs true (results not shown). Thus, ad- graph on DEAE-cellulose. sorption and elution from DEAE-Sephadex causes no detect- In order to determine whether prior separation of poly- able interconversion or alteration in the three enzymes. merases I and III on DEAE-Sephadex would affect their In a second experiment, a crude extract of yeast cells was subsequent chromatographic behavior on DEAE-cellulose, Downloaded by guest on September 29, 2021 Biochemistry: Schultz and Hall Proc. Nat. Acad. Sci. USA 73 (1976) 1031

q;70o. c,. , .

I0 Q-B 04 E~~~~~~~~~~ .2 0 ;100- 0.0

E 50-

0IO- 002

Fraction Number 0.10 0.20 0.30 040

FIG. 2. (A and B) Rechromatography of yeast RNA polymer- M Ammonium Sulfate ases I and III on DEAE-Sephadex. Yeast RNA polymerase activi- FIG. 3. Dependence of RNA polymerase I and III activity ties were initially separated on DEAE-Sephadex as in Fig. 1A. upon salt concentration. RNA polymerases I and III, purified on Fractions containing polymerase I (nos. 89-94) or polymerase III DEAE-Sephadex, were assayed in the presence of the indicated activity (nos. 135-141) were pooled and concentrated as in Materi- ammonium sulfate concentrations. Final assay conditions were als and Methods. Aliquots (0.5 ml) of the concentrated polymerase 0.055 M Tris-Cl (pH 7.9, 220), 15-20% (vol/vol) glycerol, 2 mM I or III fractions were diluted with TGED buffer to 50 mM ammo- MnCl2, 0.3 mM EDTA, 1 mM dithiothreitol, 0.1 mM [3H]UTP nium sulfate and rechromatographed in parallel on identical 0.9 x (specific activity was 150 Ci/mol), and 0.5 mM each of ATP, CTP, 16 cm columns of DEAE-Sephadex (A-25), at a flow rate of 6-7 and GTP. Native salmon sperm DNA (@--@) was present at 520 ml/hr. Fractions of 1.3 ml were collected and SO-gll aliquots assayed gg/ml or 300 ig/ml in assays with RNA polymerase I or III, respec- for RNA polymerase activity as in Materials and Methods. Re- tively. For both enzymes, poly[d(A-T)] (-----A) was present at sults for RNA polymerases I and III are presented in (A) and (B), 150 Ag/ml. Reaction mixtures were incubated 10 min at 30° and re- respectively. (C) In a separate experiment, fractions 78-97 from actions were terminated as described in Materials and Methods. the first peak of RNA polymerase activity resolved by DEAE-cel- Results for RNA polymerases I and III are shown in panels (A) and lulose chromatography (peak A of Fig. 1B) were pooled and con- (B), respectively. With native salmon sperm DNA, maximal activi- centrated as in Materials and Methods. An aliquot of the concen- ties were 337 and 342 pmol of UMP incorporated per ml for RNA trated fraction (2.0 ml) was diluted with TGED buffer to 50 mM polymerases I and III, respectively. With poly[d(A-T)], the maxi- ammonium sulfate and chromatographed on a 0.9 X 16 cm column mal activities were 122 and 177 pmol of UMP incorporated per ml of DEAE-Sephadex (A-25). Additional details were as in (A) and. for RNA polymerases I and III, respectively. (B). (D) RNA polymerases I and III were obtained in an experi- ment analogous to that shown in Fig. 1A. Fractions containing covered in this peak (Fig. 2D). Thus, RNA polymerases I polymerase I or polymerase III activity were concentrated as in Materials and Methods. Aliquots of the concentrated polymerase and III that have had prior exposure to DEAE-Sephadex I and III fractions containing equal amounts of RNA polymerase still cochromatograph on DEAE-cellulose. These results in- activity were combined and diluted with TGED buffer to 50 mM dicate that the differential chromatographic behavior of ammonium sulfate, and the mixture was chromatographed on a 0.9 RNA polymerase III on DEAE-cellulose compared to X 16 cm column of DEAE-cellulose. Other details of the chroma- DEAE-Sephadex is a property intrinsic to this enzyme and tography and assay for RNA polymerase activity were as in (A). not one dependent upon its chromatographic history. There No activity appeared in any of the flowthrough fractions of A, B, numerous with RNA C, or D (results not shown); (@--@*), RNA polymerase activity; are indications, from experiments done (--) ammonium sulfate molarity. polymerases from various animal cells and tissues (4, 5, 11- 14), that this differential chromatographic behavior on

the following rechromatography experiment was per- DEAE-cellulose compared to DEAE-Sephadex may be a formed. Yeast RNA polymerases I and III were separated by characteristic property of all class III RNA polymerases. DEAF-Sephadex chromatography. Aliquots of the two en- Influence of ionic strength on RNA polymerase zyme solutions containing equal amounts of activity were activity then mixed and applied to an analytical DEAF-cellulose col- umn (Fig. 2D). One peak of RNA polymerase activity was RNA polymerases I and III showed significant differences eluted at 0.12 M ammonium sulfate, the same salt concen- with regard to the influence of ionic strength on their re- tration at which peak A activity was eluted from DEAF-cel- spective activities, as may be seen in Fig. 3. With native lulose (see Fig. 1B). The peak fractions (nos. 36-55 in Fig. salmon sperm DNA as template, RNA polymerase I showed 2D) were then reassayed both at 40 mM and 190 mM am- a single optimum at 25 mM ammonium sulfate. In contrast, monium sulfate, since the activity measured at 40 mM salt the salt activation profile for RNA polymerase III was bi- should indicate primarily the amount of polymerase I phasic, with optima at 0.1 M and 0.25 M ammonium sulfate. present while the activity measured at 190 mM ammonium These results are similar to those reported for the class I and sulfate should be due to RNA polymerase III. Nearly equal III RNA polymerases from a variety of animal tissues (5, 11, amounts of RNA polymerase activity were measured at both 14, 22). When poly[d(A-T)] was used as template, polymer- the low and high salt concentrations, indicating that nearly ase III showed only a single optimum at 80 mM ammonium equivalent amounts of both polymerases I and III were re sulfate and polymerase I a single optimum at 10 mM ammo- Downloaded by guest on September 29, 2021 1032 Biochemistry: Schultz and Hall Proc. Nat. Acad. Sci. USA 73 (1976) less than 35% inhibited at 2.4 mg of a-amanitin per ml. Sim- ilar results (not shown) for the yeast enzyme III have also been obtained using denatured salmon sperm DNA as tem- plate. DISCUSSION Our studies of salt dependence of enzyme activity, template specificity, and chromatographic behavior on DEAE-substi- 50 tuted cellulose and Sephadex columns all support the con- cept of a homology between yeast and vertebrate RNA po- lymerases of the form: yeast I vertebrate I, yeast II- XA vertebrate II, and yeast III = vertebrate III. Like vertebrate class I polymerases, yeast polymerase I elutes early from both anion exchangers and has a salt optimum of less than 10-2 10- 100 102 103 104 0.07 M (NH4)2SO4 (3-5, 7, 11-15, 18, 22, 29). Like verte- Qa-AMANITIN 9A/ml) brate class II RNA polymerases, yeast RNA polymerase II is FIG. 4. Sensitivity of yeast RNA polymerases I, II, and III, pu- eluted from both DEAE-Sephadex and DEAE-cellulose at rified on DEAE-Sephadex, to a-amanitin. Assay conditions were approximately 0.22 M (NH4)2SO4 and is much more active 0.05 M Tris-Cl (pH 7.9, 220), 5% (vol/vol) glycerol, 1.6 mM MnCl2, on denatured than on native DNA templates (3-5, 7, 12, 13, 1 mM dithiothreitol, 0.05 mM [3HJUTP (specific activity was 300 16, 18). Like vertebrate class III RNA polymerases (11-14, Ci/mol), and 0.5 mM each of ATP, CTP, and GTP. Heat-dena- 28), yeast enzyme III cochromatographs with polymerase I tured salmon sperm DNA was present at 100 /g/ml in assays with on DEAE-cellulose, but is eluted at much higher ionic RNA polymerases I and II. Poly[d(A-T)] was present at 50 ;ig/ml in assays with polymerase III. Final ammonium sulfate concentra- strength than enzyme I on DEAE-Sephadex. The yeast and tions in the assay were 30 mM, 60 mM, and 100 mM ammonium vertebrate enzymes III are also similar as regards their char- sulfate for RNA polymerases I, II, and III, respectively. Appropri- acteristic double salt optima for activity on native DNA (5, ate amounts of a-amanitin were added to the substrate solution 7, 11, 14, 22) and their high activity on a poly[d(I-C)] temr- prior to the initiation of the reactions by addition of enzyme. Reac- plate (R. G. Roeder, personal communication). The analo- tion mixtures were incubated 5 min at 300. Other details were as in gies that these similar catalytic properties suggest are rein- Materials and Methods. Maximal activities were 268, 123, and 153 pmol of UMP incorporated per ml for RNA polymerases I, II, and forced by the comparisons that have been made between the III, respectively. (A *) Polymerase I activity; (0 *) poly- structures of vertebrate and yeast enzymes I merase II activity; (-u*) polymerase III activity. and II (3, 15) and enzymes III (15). The different patterns of a-amanitin resistance between nium sulfate. Schwartz et al. (11) reported qualitatively sim- yeast and mammalian RNA polymerases contrast sharply ilar results for the class I and class III RNA polymerases with the similarities that exist between these two groups of from mouse plasmacytoma. enzymes with regard to other properties. These differences are of two types. First, there is a generally higher level of re- Activity with a synthetic DNA template sistance of yeast enzymes to a-amanitin, as exemplified by Yeast RNA polymerases I and III differ greatly in their abili- the midpoints of the inhibition curves of yeast and verte- ty to transcribe the alternating copolymer, poly[d(I-C)]. brate enzymes II, which are 0.01 ,ug/ml and 1.0,g/ml, re- Polymerase I shows about a 25-fold greater activity with na- spectively (3, 7, 9, 17). Second, the relative a-amanitin sensi- tive salmon sperm DNA than with poly[d(I-C)]. On the tivities of enzymes I and III are reversed. Yeast RNA poly- other hand, polymerase III shows nearly a 2-fold preference merase I is 50% inhibited by 600 jtg/ml of a-amanitin (Fig. for poly[d(I-C)] compared to native salmon sperm DNA (re- 4), a concentration at which class I vertebrate RNA poly- sults not shown). The RNA polymerases from Xenopus lae- merases are fully resistant (11-14, 25-28). The a-amanitin tvs show qualitatively similar, though less dramatic, differ- sensitivity of yeast RNA polymerase III also appears anoma- ences in their ability to transcribe poly[d(I-C)] (Robert G. lous when compared to the vertebrate polymerase results. Roeder, personal communication). The X. laevis class I RNA Whereas the class III RNA polymerases of vertebrate cells polymerases are more active with native X. laevts DNA than are 50% inhibited by 10-40 Ag/ml of a-amanitin (11-14, with poly[d(I-C)]. In contrast, the X. laevis class III RNA po- 25-28), yeast RNA polymerase III is 95% resistant to 600 lymerases show several-fold greater activity with poly[d(I- gg/ml of a-amanitin and 65% resistant to 2.4 mg/ml (Fig. C)] as template than with native DNA (R. G. Roeder, per- 4). Yeast RNA polymerase III may indeed be completely re- sonal communication). sistant to the specific inhibitory effect of a-amanitin, since nonspecific effects might be expected at this level of drug. Sensitivity of yeast RNA polymerases to a-amanitin Roeder and Gage have found that RNA polymerase III of Yeast RNA polymerase II is the most sensitive of the three the insect Bombyx mori, like yeast RNA polymerase III, is RNA polymerases to inhibition by a-amanitin (Fig. 4) (7, 8, virtually completely resistant to inhibition by a-amanitin 17, 18). It is inhibited by 50% at 1.0 ,g/ml and totally inhib- (14). ited at 50 gg of a-amanitin per ml. In contrast to the find- The results of a-amanitin inhibition studies on yeast and ings for the vertebrate class I RNA polymerases (11-14, 25- Bombyx RNA polymerase illustrate the difficulties inherent 28), yeast RNA polymerase I can be completely inhibited by in applying to lower the classification schemes high concentrations of a-amanitin (50% inhibition at 600 that have been devised for vertebrate RNA polymerases. Atg/ml). On the other hand, yeast RNA polymerase III is less The Roeder-Rutter nomenclature (class I, II, and III RNA sensitive to a-amanitin than either yeast polymerase I (Fig. polymerases) uses as classification criteria the catalytic and 4) or the vertebrate class III polymerases (11-14, 25-28). chromatographic properties of the various enzymes (1, 2), Yeast polymerase III is only 5% inhibited at 600 gg/ml and particularly salt dependence, while the Chambon nomencla- Downloaded by guest on September 29, 2021 Biochemistry: Schultz and Hall Proc. Nat. Acad. Sci. USA 73 (1976) 1033 in The Enzymes, ed. Boyer, P. D. (Aca- is based upon the 3. Chambon, P. (1974) ture (class A, B, and C RNA polymerases) York), pp. 261-331. resistance of each RNA polymerase (4). demic Press, New level of a-amanitin 4. Chambon, P. (1975) Annu. Rev. Biochem. 44,613-38. For vertebrate RNA polymerases, these two classification 5. Sergeant, A. & Krsmanovic, V. (1973) FEBS Lett. 35, 331- systems are entirely equivalent, since every class A RNA 335. polymerase (completely a-amanitin resistant) is also class I, 6. Dezelee, S., Sentenac, A. & Fromageot, P. (1972) FEBS Lett. every RNA polymerase B (highly a-amanitin sensitive) is 21, 1-6. also class II, and every RNA polymerase C (slightly a-aman- 7. Adman, R., Schultz, L. D. & Hall, B. D. (1972) Proc. Nat. itin sensitive) is also class III. However, this correspondence Acad. Sci. USA 69, 1702-1706. a-amanitin resistance and other enzyme properties 8. Tipper, D. J. (1973) J. Bacteriol. 116,245-256. between E. (1971) FEBS Lett. does not hold for the RNA polymerases from yeast and 9. Ponta, H., Ponta, U. & Wintersberger, RNA I, for example, which has 18,204-208. Bombyx. Yeast polymerase 10. Sklar, V. E. F., Schwartz, L. B. & Roeder, R. G. (1975) Proc. been designated RNA polymerase A by Sentenac and co- Nat. Acad. Sci. USA 72,348-352. workers (6, 17), actually has the level of a-amanitin resis- 11. Schwartz, L. B., Sklar, V. E. F., Jaehning, J. A., Weinmann, R. tance characteristic of a class C enzyme (Fig. 4; and ref. 4). & Roeder, R. G. (1974) J. Biol. Chem. 249,5889-5897. Classification ambiguities of this type, which are most seri- 12. Seifart, K. H. & Benecke, B. J. (1975) Eur. J. Biochem. 53, ous for the RNA polymerases from simple eukaryotes, un- 293-300. derscore the need for an RNA polymerase nomenclature 13. Austoker, J. L., Beebee, T. J. C., Chesterton, C. J. & Butter- based upon enzyme structure and function (4, 10, 15) rather worth, P. H. W. (1974) Cell 3, 227-234. F. & R. G. (1975) Fed. Proc., Fed. Am. than upon one or a few properties of the isolated enzymes. 14. Sklar, V. E. Roeder, Soc. Exp. Biol. 34,2448. The basis for a functional classification of vertebrate cell 15. Valenzuela, P., Hager, G. L., Weinberg, F. & Rutter, W. J. RNA polypnerases was provided by the experiments of differ- (1976) Proc. Nat. Acad. Sci. USA 73, 1024-1028. Weinmann and Roeder. These authors exploited the 16. Dezelee, S. & Sentenac, A. (1973) Eur. J. Biochem. 34, 41-52. ing a-amanitin sensitivities of class I, II, and III RNA poly- 17. Buhler, J.-M., Sentenac, A. & Fromageot, P. (1974) J. Biol. merases to identify the RNA transcripts made by each class Chem. 249,5963-5970. of enzyme in isolated cell nuclei. In mouse nuclei, 5S and 4S 18. Ponta, H., Ponta, U. & Wintersberger, E. (1972) Eur. J. Bio- precursor RNA synthesis exhibited the a-amanitin sensitivity chem. 29, 110-118. characteristic of class III RNA polymerases (26), while in ad- 19. Hartwell, L. H. (1967) J. Bacterlol. 93, 1662-1670. enovirus-infected human cell nuclei, viral 5.5S and cellular 20. Bhargava, M. M. & Halvorson, H. 0. (1971) J. Cell Biol. 49, 5S RNAs were shown to be made by RNA polymerase III 423-429. RNA II (27). Now that tran- 21. Mans, R. J. & Novelli, G. D. (1961) Arch. Biochem. Biophys. and viral mRNA by polymerase 94,48-53. can be isolated scriptionally active yeast nuclei reproducibly 22. Roeder, R. G. (1974) J. Biol. Chem. 249, 241-248. (refs. 30 and 31; L. D. Schultz, manuscript in preparation), 23. Sebastian, J., Bhargava, M. M. & Halvorson, H. 0. (1973) J. differential a-amanitin inhibition of RNA synthesis in isolat- Bactetiol. 114, 1-6. ed yeast nuclei can be used to identify the function of each 24. Brogt, Th. M. & Planta, R. J. (1972) FEBS Lett. 20, 47-52. yeast RNA polymerase. By examining the RNA products 25. Weil, P. A. & Blatti, S. P. (1975) Biochemistry 14, 1636-1642. made in yeast nuclei under increasing degrees of a-amanitin 26. Weinmann, R. & Roeder, R. G. (1974) Proc. Nat. Acad. Sci. inhibition, we are determining which yeast RNA polymer- USA 71, 1790-1794. & Roeder, R. G. (1974) Proc. ase makes each class of RNA. 27. Weinmann, R., Raskas, H. J. Nat. Acad. Scl. USA 71, 3426-3430. We thank Dr. Robert G. Roeder for helpful discussions and for 28. Beebee, T. J. C. & Butterworth, P. H. W. (1974) FEBS Lett. informing us about his unpublished results. This research was sup- 47,304-306. ported by Research Grant (GM-11895) and Biochemistry Training 29. Blatti, S. P., Ingles, C. J., Lindell, T. J., Morris, P. W., Weaver, Grant (GM-00052) from the National Institutes of Health. R. F., Weinberg, F. & Rutter, W. J. (1970) Cold Spring Har- bor Symp. Quant. Biol. 35, 649-657. 1. Roeder, R. G. (1969) Ph.D. Dissertation, University of Wash- 30. Lohr, D. & Van Holde, K. E. (1975) Science 188, 165-166. ington. 31. Wintersberger, U., Smith, P. & Letnansky, K. (1973) Eur. J. 2. Roeder, R. G. & Rutter, W. J. (1969) Nature 224, 234-237. Biochem. 33, 123-130. Downloaded by guest on September 29, 2021