The DNA-directed RNA polymerases of soybean

Bioehimiea et Biophysica Aeta, 294 (1973) 227-235 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 97526

T H E DNA-DIRECTED RNA POLYMERASES OF SOYBEAN

PAUL A. HORGEN* AND JOE L. KEY Department of Botany, University of Georgia, Athens, Ga. 3060± (U.S.A.)

(Received July 3Ist, 1972)

SUMMARY

Four cellular RNA polymerases have been partially purified from hypocotyls of soybean (Glycine max). Enzyme isolation from nuclear preparations indicate the DEAE polymerases I and I I are localized within the nucleus. Inhibitor studies showed that DEAE polymerase I I was preferentially inhibited by ~-amanitin, DEAE peaks I I I and IV were sensitive to rifampin, whereas all of the polymerases were insensitive to cycloheximide. The DEAE polymerase I was most active in the presence of Mn z+, whereas the DEAE polymerase 21 was more active in the presence of Mg 2+. DNA stimulation and dependency on the four riboside triphosphates were shown to be characteristic of all four polymerase fractions. KOH hydrolysis and ribonuclease digestion indicate that the product was a polyribonucleotide. RNA polymerase I was most effective with native soybean nuclear DNA, whereas RNA polymerase 21 preferred denatured soybean nuclear DNA. The kinetics of the polymerizing reactions were linear for IO min for both nuclear enzymes at 30 °C. Further purification was a t tempted using glycerol gradient centrifugation and chromatography on phosphocellulose. Glycerol gradient centrifugation of the nuclear polymerases resolved each into two components. Phosphocellulose chromatography of polymerase I I resulted in two components differing in their sensitivity to ~-amanitin.

INTRODUCTION

Multiple RNA polymerases exist in a large number of eukaryotic organisms. More than one enzyme exists in the nuclei of animal cells 1-3, fungal cells 4-6, and the cells of higher plants 7,8. At least one enzyme (polymerase I) is compartmentalized within the nucleolus ~,9-u and is presumably involved in the synthesis of ribosomal RNA in vivo. This enzyme is specifically inhibited by cycloheximide in primitive aquatic fungi 4,12. There also exists at least one polymerase (polymerase II) in the non-nucleolar nucleoplasm 2,~,~3 which is apparently involved in the synthesis of DNA-like RNA in vivo. x-Amanitin, a poisonous toxin from the Basidiomycete Amanita phaltoides, preferentially inhibits the nucleoplasmic enzyme (polymerase

Abbreviation: PMSF, phenylmethyl sulfonylfluoride. Enzyme: DNA-dependent RNA polymerase or nucleoside triphosphate; RNA nucleotidyl-

transferase (EC 2.7.7.6 ). * Present address: Department of Botany, University of Toronto, Erindale College, Missis-

auga, Ontario, Canada.

228 p . A . H O R G E N , J . L. K E Y

I I ) in all eukaryotes studied. A third nuclear enzyme of unknown function also has been reported from certain eukaryotic tissues 1'5'6'1z. Organelle-specific RNA polymerases are present in mitochondria 14 ~8 and in chloroplasts 19.

We have examined the properties of the cellular RNA polymerases of soybean hypocotyl, particularly those enzymes compartmentalized within the nucleus. This is the initial step leading to studies of the effects of auxins on the regulation of trans- cription in these tissues. This s tudy revealed multiple RNA synthesizing enzymes in soybean. The properties of the enzymes in respect to metal ion concentrations, template specificity and sensitivity to polymerase inhibitors were examined and dis- cussed in comparison with the properties of the RNA polymerases of other eukaryotes. The results of a t tempts to further purify and stabilize the activities of the soybean enzymes are also reported.

M A T E R I A L S A N D M E T H O D S

Isolation o/cellular RNA polymerases Etiolated seedlings of soybean (Glycine max, var. Hawkeye 63) were grown

under moist layers of vermiculite for 72 h. 3-cm sections of the hypocotyl were remo- ved directly below the a t tachment of the cotyledons and placed in homogenizing buffer: o.oi M Tris-HC1 (pH 7.9), I .o M sucrose, 5 mM MgC12, 0.5 mM dithiothreitol and 0.5 mM phenylmethyl sulfonylfluoride. When indicated, 0.2 °//o bovine serum albumin was added to the homogenizing buffer and all subsequent buffers. Approxi- mately 20-30 g fresh wt of tissue were homogenized in 50 ml of the buffer for 25 s at high speed in a Willems Polytron. The homogenate was then filtered through miracloth and brought to a concentration of 0.2 M (NH4)2SO 4 (with 4 M (NH4)2SO 4 brought to a pH of 7.9 with NH~OH). The mixture was either rehomogenized in a ground glass tissue grinder or was sonicated in I2.5-ml aliquots for 15 s in a Bronwill Biosonik (setting 8). The suspension (Fraction A) was then rapidly mixed with 2 vol. of 0.05 M Tris-HC1 (pH 7.9)-25 °." o glycerol- 5 mM MgC12-o.I mM EDTA 0.5 mM dithiothreitol buffer 9 containing 0.5 mM PMSF. The suspension was centrifuged for I h in a Sorvall centrifuge at 31 ooo × g. The supernatant (Fraction B) was brought to near saturation with (NH4)2SO4 by the addition of 0.42 g of solid (NH4) 2- S Q per ml of solution 9. The precipitate was collected by centrifugation for I h in a Sorvall centrituge at 31 ooo x g and resuspended in 0.05 M Tris-HC1 (pH 7.9)-25 °, o glycerol- 5 mM MgC12-o.I mM EDTA 0.5 mM dithiothreitol 9 containing 0. 5 mM PMSF (Fraction C). After dialysis for 16 h against 0.05 M Tris HC1 (pH 7.9)-25 3'o glycerol 5 mM MgC12 o.I mM EDTA-o.5 mM dithiothreitol containing 0.5 mM PMSF the dialysate was centrifuged at 31 ooo × g for 20 rain. The supernatant (Fraction D) contained the crude soluble cellular RNA polymerases. The temperature was main- tained at 4 ± I °C during the isolation procedure.

Isolation o/ a nuclear enriched/raction Approximately 4o g of soybean hypocotyl were cut as previously described

and suspended in nuclear homogenizing buffer: 20 mM potassium phosphate (pH 6.5), -5 mM MgC12- 7 mM KCI-o. 5 M sucrose 0. 5 mM dithiothreitol-o.5 mM PMSF. The tissue was homogenized with a Willems Polytron (setting 7 for 25 s) and filtered

SOYBEAN POLYMERASES 229

through miracloth. The filtrate was centrifuged at 77oo × g for 2o min and resultant supernatant discarded. The nuclear material was scraped off the starch pellet and resuspended in the nuclear homogenizing buffer. The suspension was centrifuged for 20 min at 7000 × g and the nuclear material again scraped off the starch pellet and subjected to another wash at 7000 × g for 20 min. The nuclear material of the precipitate was resuspended in the nuclear homogenizing buffer and layered on 25 ml of 1. 7 M sucrose containing 20 mM potassium phosphate (pH 6.5), 5 mM MgC12, 7 mM KCI, 0.5 mM dithiothreitol and 0.5 mM PMSF. The upper 1/3 of the centrifuge tube was gently swirled, and the material was centrifuged for 9 ° min at 22 ooo rev./min in an SW 25.1 rotor. The resulting pellet was examined microscopically and was found to contain whole nuclei, broken nuclei and starch grains. The enriched nuclear pellet was then suspended in the cellular homogenizing buffer and was subjected to high salt sonication, (NH4)~SO * precipitation and dialysis as described previously. The dialysate was centrifuged as before, and the resulting supernatant contained the crude soluble nuclear RNA polymerases.

D EA E-cellulose chromatography 12 ml of the crude cellular RNA polymerases (3o-6o mg protein) or 5 ml of the

crude nuclear RNA polymerases (15-25 mg protein) were layered on a DEAE-cellulose column (I cm × IO cm). Chromatography on DEAE followed the procedure of Mertelsmann and Matthaei ~° with 0.5 mM dithiothreitol (instead of mercaptoethanol) and 0. 5 mM PMSF added to the chromatography buffer. The enzymes were eluted with a linear gradient of (NH4)2SO * from 50 to 500 mM. I-ml fractions were collected at 4 + I °C. Aliquots of the eluates were immediately made to I mg/ml bovine serum albumin in an a t tempt to stabilize enzyme activity.

Assay ]or R N A polymerase activity RNA polymerase activity was determined according to the procedure of Stout

and Mans 21. Assays were run for 5 min at 30 °C with salmon sperm DNA normally used as the template. Either [3H~ATP or [3HIUTP was incorporated as the labeled monophosphate into trichloroacetic acid-insoluble polynucleotides. The precipitated product was collected on Whatman GFA glass fiber filters, washed with Hokins solution *S, dried and counted by liquid scintillation. Protein determinations were made by A2s 0 nm/A2e 0 nm ratios.

Glycerol gradient centri]ugation and chromatography on phosphocellulose The peak fractions of the nuclear RNA polymerases were pooled and dialyzed

against 0.05 M Tris-HC1 (pH 8.o), 0.5 mM dithiothreitol, 0. 5 mM PMSF, 5 % (v/v) glycerol for 3 h at 4 ~ I °C. The enzyme preparation (I ml) was then layered on a 25-ml linear gradient of lO-3O % glycerol in 0.05 M Tris-HC1 (pH 8.o), 0.5 mM dithiothreitol, 0.5 mM PMSF. The gradients were centrifuged for 13 h at 22 ooo rev./min in a Spinco SW 25.1 Rotor. A marker, Escherichia coli fl-galactosidase (16 S), was centrifuged on a parallel gradient.

Phosphocellulose chromatography of RNA polymerases I and I I was carried out according to the procedures of Weaver et al. *s.

Chemicals The unlabeled nucleoside triphosphates, salmon sperm DNA, Micrococcus

2 3 0 P . A . HORGEN, J. I,. KEY

lysodeikticus DNA, E. coli fl-galactosidase, dithiothreitol, PMSF, and bovine serum albumin were purchased from Sigma Chemical Co., St. Louis. ~-Amanitin was purchased from Henley and Co., N. Y.; rifampin, cycloheximide (acetidione), DEAE- cellulose (Cellex D, Bio-Rad), and phosphocellulose (Cellex P, Bio-Rad) from Cal- biochem, Los Angeles. The [3HIATP and [3H]UTP were obtained from Schwarz/ Mann, Orangeburg, N. Y. Enzyme grade (NH4)2SO 4 was obtained from Nutritional Biochemicals, Cleveland. All other chemicals used were reagent grade.

Soybean DNA was a gift of Dr Alan Jaworski, Botany Department , University of Geolgia.

RESULTS

No RNA polymerase activity could be detected in Fractions A, B, C, or D of the purification procedure. Only after DEAE-cellulose chromatography was any enzyme act ivi ty measurable. The polymerase isolation procedure used by Horgen and Griffin 4 for Blastocladiella emersonii did not produce soluble multiple polymerases from soybean hypocotyl. Active preparations of soybean RNA polymerases were obtained repeatably only after PMSF was added to the buffers of the procedure described.

Four peaks of RNA polymerase activity were eluted from DEAE-cellulose (Fig. IA) when polymerases were solubilized from the cellular homogenate. Peak I was eluted at approximately o.I 5 M (NH4)2SO 4, Peak I I at o.28 M, Peak I I I at o.33 M and Peak IV at o.4I M (NH4)2SO 4. In an a t tempt to determine which of these enzymes were of nuclear origin, a nuclear enriched fraction was obtained and the RNA polymerases solubilized. Fig. IB shows the DEAE elution pat tern from the nuclear material. Two major fractions of RNA polymerase activity were observed. These two activities were eluted at approximately the same (NH4)2SO 4 concentration as Peak I and Peak I I from the whole cellular homogenate. Rechromatography of enzymes I and I I (Fig. IC) suggested no evidence of dissociation of the individual complexes or interconvertibility of the two forms on DEAE-cellulose. Each of the polymerase activities showed a marked stimulation with added salmon sperm DNA and a depen- dence on the presence of the four riboside triphosphates (Table I). The product of the enzymatic reactions was sensitive to ribonuclease digestion and KOH hydrolysis (Table I).

The effects of various polymerase inhibitors on the in vitro activities of the soybean polymerases were measured. Cycloheximide (Ioo or 2oo #g/ml) had no inhibi- tory effect on any of the polymerases of soybean. ~-Amanitin preferentially inhibited the Peak I I enzyme but had no effect on the other polymerases. Fig. 2 shows the effect of varying the concentration of ~-amanitin on the nuclear polymerases of soybean. 5o % inhibition of the Peak I I enzyme was observed with 8 #g/ml of the toxin. Enzyme activity trom Peaks I I I and IV were both completely inhibited by I5o/~g/ml rifampin, whereas enzymes I and I I were not affected.

The effect of varying the concentration of divalent cations upon activity of the nuclear enzymes from DEAE Peaks I and I I was measured. The Peak I enzyme exhibited optimal activity at 5 mM Mn 2+ and 2o mM Mg 2+, being most active in the presence of Mn 2+ (Fig. 3). The Peak I I enzyme showed optimal activity at 5 mM Mn 2+ and 2o mM Mg 2+, being most active in the presence of Mg 2+ (Fig. 3). Both nuclear polymerases showed linear kinetics of incorporation for Io min at 3 ° °C.


A

II 500

1.5 ~ 0.5 2500 1.0 0.3 0.5

0.1 0 0

B II

o o.3

0.1 < ~ 0.5

0.3

0.1

1~ I 0.5 0.3

0.1 0 4 8 12 16 20 24

Fraction No. Fig. I. DEAE-cel lulose ch romatography of soybean R N A polymerases . (A) Chromatography of polymerases from crude cellular extract . The column was washed with 15o ml of buffer con- ta ining IO mM Tris-HC1 (pH 7.8), 5 ° mM (NH4)~SO~, 6 mM MgC12, I mM potass ium EDTA, 3 ° ?/o (v/v) glycerol, 0. 5 mM dithiothrei tol , 0. 5 mM PMSF. React ion mixtures as in Table I. The temperature during chromatography was mainta ined at 4 ± I °C. (B) D E A E chromatography of polymerases from nuclear enriched fraction. DEAE-cel lulose column ch roma tography followed by assay was as described for A. (C) Reehroma tography of the nuclear R N A polymerases . Ap- prox imate ly 3 ml of either Peak I or Peak II (2- 4 mg protein) was chromatographed and assayed as described above.

The ability of soybean nuclear RNA polymerases to utilize templates from a variety of sources is shown in Fig. 4. Native soybean DNA was the most effective template for enzyme I activity (Fig. 4A). Eukaryotic DNA proved to be a better template than prokaryotic DNA, enzyme I preferred native to denatured template in all cases examined. The DEAE enzyme II fraction was most effective utilizing denatured soybean DNA as a template (Fig. 4B). Again, eukaryotic DNA was a better template than prokaryotic DNA; enzyme II more effectively utilized denatured than native template in all cases examined.

Results of attempts to further purify the nuclear RNA polymerases on glycerol gradients for both DEAE enzyme I and II resolved two peaks of activity for each DEAE enzyme. One peak of activity had a sedimentation value close to that ot fl- galactosidase (16 S), whereas the other peak of activity sedimented much faster. In

232 P .A . HORGEN, J . L . KEY

TABLE I

A N A L Y S I S OF S O Y B E A N C E L L U L A R RNA P O L Y M E R A S E S

A complete reaction mixture contained Tris-HC1, magnesium acetate, dithiothreitol , (NH4)2SO ~ and DNA (salmon sperm) according to Stout and Mans 21. UTP, GTP, and CTP (o.5/~mole/ reaction mixture), Iot l l enzyme preparation (6oo-9oo/,g protein as determined by d , s 0 ,m/ A a00 n m ) , 0"45 Fmole ATP and ~3HIATP (2.5/~Ci/reaction mixture, 2o. 5 Ci/mmole) were also added. Reaction was run for 5 rain at 3 ° °C and stopped by the addition of ice-cold io % trichloroacetic acid containing o.8 mM Na, P~O 7. The RNA precipitate was collected on glass fiber filters, washed with 5 ml Hokins solutionS% dried and counted by liquid scintilation. Specific activity for con- trols ranged from 4O-lOO pmoles AMP incorporated/min per mg protein.

C o n d i t i o n s ° o A c t i v i t y o.f control

P e a k 1 P e a k I1 P e a k I I I P e a k l t "

Complete i oo.o t oo.o i oo.o i oo.o - -DNA 3.8 5.6 4.2 2.8

UTP, G T P , CTP o,o o,o o,o o,o +0. 3 M KOH 14.o o.o ~.2 3.o + ribonuclease (Ioo pg/ml) ~3.o 12. 7 i i .3 1°-9

100

80

c_)

40

1I

O0 ]b 20 30 40 50 ug of ~-amanit in

Fig. 2. "/'he effect of ~-amanitin concentration on the activity of soybean nuclear RNA polymerases. Assay procedure as in Table I. ~-Amanitin was added to the reaction mixture before the addition of the enzyme.

t h e s e e x p e r i m e n t s , bov ine s e r u m a l b u m i n (o.2 % ) was a d d e d to all bu f fe r s to he lp

s t ab i l i ze t h e v e r y lab i le n u c l e a r p o l y m e r a s e s .

R e s u l t s ot c h r o m a t o g r a p h y on p h o s p h o c e l l u l o s e of e n z y m e I I a re s h o w n in

F ig . 5. D E A E e n z y m e I [ w a s r e s o l v e d i n t o t w o f u r t h e r a c t i v i t i e s u p o n p h o s p h o c e l l u -

lose c h r o m a t o g r a p h y . T h e s e t w o ac t iv i t i e s d i f fe r in t h e i r s e n s i t i v i t y to ~ - a m a n i t i n .

P h o s p h o c e l l u l o s e I I a w a s i n h i b i t e d 80 % b y 25 # g / m l ~ - a m a n i t i n , w h e r e a s P e a k I I b

w a s i n h i b i t e d o n l y 35 % b y t h e s a m e c o n c e n t r a t i o n of t h e t o x i n .


225 II Iii /%

0"1 I 2+

~ I¢ ', 2 ~r, ' ,

c_ 13-

k .........~ Mg 2 +

25

.E 20

~15

L 10

.c 5 E

~ o o ~45

O E 30

15

0 ' ;~0 ' 4b ' go 0 Concentration (rnM)

A

i" ~ S B n

--- ~ - • SSn

j / " ~, OSBd 1 7 ,,,/t ° . . . . ~ SSd

' 1~) ' 20 ' 30 ' 4 0

~ S B d

, ~ SBn

rA ............ ~ - -.~-~- ........ SSn I ~ o ~ . ~ - - ° ~ ; ' - - 4 MLd f / - ~ " , " - . . . . . . . . . . • MLn

' g ' 6 ' lg ' 5o DNA ( jag )

Fig. 3. Effect of divalent cation concentration on the activi ty of soybean nuclear polymerases. Assay as in Table I with either 2.5 #Ci [3H]ATP or 2. 5/~Ci [sH]UTP (13 Ci/mmole) added to the reaction mixture. NMP, nucleoside monophosphate.

Fig. 4. Template specificity of soybean nuclear RNA polymerases. Assay conditions as in Table I using [aH] UTP (I 3 Ci/mmole). SBn, soybean native; SSn, salmon sperm native; MLn, Micrococcus lysodeikticus native; SBd, soybean denatured; SSd, salmon sperm denatured; MLd, Micrococcus lysodeikticus denatured. DNA was denatured by heating for io min at ioo °C and then plunging the test tube into an ice bucket and using the DNA immediately as a template for the reaction.

.c

~ 1 5 0

g L

8mc

. . . . . i , i . . . . .

4 8 12 16 20 24 Fraction No.

400

E

Fig. 5. Phosphocellulose chromatography of DEAE RNA polymerase II. Assay conditions are as in Table I only using [sH]UTP (13 Ci/mmole). For this experiment 0.2 % bovine serum albumin was added to all buffers during the solubilization and chromatography of the enzyme activities.

234 P . A . HORGEN, J . L . KEY

DISCUSSION

Four distinct RNA polymerases have been isolated from cellular homogenates of soybean hypocotyl. Two of these enzymes are of nuclear origin as demonstrated by solubilization of the polymerases from nuclear enriched preparations. The two DEAE-fractionated nuclear activities of soybean are consistent with results from other eukaryotic organisms 2'3'14 and with other plants 7'8.

The sensitivity of enzyme II to e-amanitin indicates that this activity corresp- onds to the nucleoplasmic enzyme, RNA polymerase II, found in other eukaryotes 2 4.8. The DEAE RNA polymerase I has been demonstrated as being nucleolar 2,9,1° in animals and has been inferred as being nucleolar in fungi 4'a'1~. Based on its location in the nucleus, insensitivity to e-amanitin, and its chromatographic behavior on DEAE, the enzyme I activity of soybean is the most likely candidate for the nucleolar RNA polymerase I. Its insensitivity to cycloheximide suggests that the DEAE polymerase I of aquatic fungi 4'12 differs in some properties from the RNA polymerase I of higher plants.

Since enzymes III and IV of soybean exhibited sensitivity to rifampin, they may represent organelle polymerases solubilized from the crude cellular extracts 14.18. The possibility also exists, although less likely, that one or both of the rifampin- sensitive activities may be the result of bacterial contamination.

RNA polymerase I of soybean responded to concentrations of divalent cations similar to the RNA polymerase I of maize s. However, unlike maize polymerase I s, but like the enzyme in B. emersonii ~, higher animal tissues 1,24 and coconut endosperm 25, polymerase I from soybean showed optimal activity in the presence of Mn 2+. RNA polymerase II from soybean was similar to the maize polymerase II s in its response to divalent cation concentrations. Maize s, coconut endosperm 25 and soybean polymerase II exhibited optimal enzymatic activity in the presence of Mg 2+. This is in contrast to observations with animals TM and fungi 4'5 where again Mn 2+ gave higher rates of incorporation.

Experiments undertaken to determine the template specificity of the soybean nuclear polymerases (Fig. 4) indicated that the homologous soybean nuclear DNA was most efficiently utilized. Polymerase I preferred native to denatured DNA and eukaryotie to prokaryotic DNA in all cases. These results are generally in agreement with those from other eukaryotic organisms 5,s'2a'26. Polymerase II showed higher activity with a denatured template and with eukaryotic DNA. RNA polymerase II of soybean is therefore similar in its template requirement to other eukaryotic nueleo- plasmic RNA polymerases 5's'~4'27.

Attempts to further purify RNA polymerase II of soybean on phosphoeellu- lose columns resolved two peaks of activity differing in their sensitivity to ~-amanitin. These results suggest that the DEAE polymerase II of soybean consists of two activities; one analogous to the nucleoplasmic RNA polymerase II found in other eukaryotes ~'13, and another activity much less sensitive to e-amanitin. Because of the extreme instability of RNA polymerase I from soybean and maize 8, attempts to further purify the enzyme on phosphocellulose columns have been generally unsuccessful.

Glycerol gradient centrifugation of the DEAE nuclear enzymes gave two activity regions for each enzyme. One activity sedimented very close to the I6-S E. coli fl-galactosidase marker, while the other activity was located much further


down the gradient. The slower sediment ing ac t iv i ty appeared similar in size to the activities observed for other eukaryot ic polymerases I and I I 8,~7. The faster sedi- men t ing activities may be the result of aggregation 2s.

At t empts to s tudy fur ther the properties of the phosphocellulose activities and the glycerol gradient enzymes have been severely hampered by the extreme labile n a t m e of the p lant polymerases. Once the enzymes have been eluted from DEAE, they are very unstable. At t empts to improve s tabi l i ty have been only modera te ly successful. We are present ly a t t empt ing to f ind methods to stabilize the soybean enzymes, to obta in further purification and characterization.

ACKNOWLEDGEMENTS

This research was supported by Public Heal th Service Research Grant CAII624 from the Nat ional Cancer Ins t i tu te to J. L. K. The technical assistance of I lona Horgen, Martha Ross and Hwei-shing Lin was great ly appreciated.

REFERENCES

I Roeder, R. G. and Rutter, W. J. (1969) Nature 224, 234-237 2 Jacob, S. T., Sajdel, E. M. and Munro, H. M. (197 o) Biochem. Biophys. Res. Commun. 38,

765-77 ° 3 Kedinger, C., Gniazdowski, M., Mandel, Jr, J. L., Gissinger, F. and Chambon, P. (197 o)

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1194-12oo 8 Strain, G. C., Mullinix, K. P. and Bogorad, L. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2647-2651 9 Roeder, R. G. and Rutter, W. J. (197 o) Proc. Natl. Acad. Sci. U.S. 65, 675-682

io Roeder, R. G. and Rutter, W. J. (197 o) Biochemistry 9, 2543-2553 I i Tocchini-Valentini, G. P. and Crippa, M. (197 o) Nature 228, 993-995 12 Timberlake, W., McDowell, L. and Griffin, D. (1972) Biochem. Biophys. Res. Commun. 46,

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(197 o) Cold Spring Harbor Symp. Quant. Biol. 35, 693-707 25 Mondal, H., Mandal, R. and Biswas, B. (1972) Eur. J. Biochem. 25, 463-47 o 26 Chesterton, C. J. and Butterworth, P. (1971) Eur. J. Biochem. 19, 232-241 27 Stout, E. R. and Mans, R. J. (1968) Plant Physiol. 43, 405-4 lo 28 Hayes, D. (1967) Annu. Rev. Microbiol. 21, 369-382

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The DNA-directed RNA polymerases of soybean