Properties of a ribonucleic acid synthesizing system in cell-free extracts of tobacco leaves

BIOCHIMICA ET BIOPHYSICA ACTA

205

BBA 95071

PROPERTIES

O F A R I B O N U C L E I C ACID S Y N T H E S I Z I N G

IN CELL-FREE

SYSTEM

E X T R A C T S O F TOBACCO L E A V E S

J. SEMAL*, D. SPENCER**, Y. T. KIM AND S. G. WILDMAN

Department of Botany and Plant Biochemistry, University of California, Los Angeles, Calif. (U.S.A.) (Received March 4th, i964)

SUMMARY

The capacity of tobacco-leaf extracts to incorporate ATP and GTP into a coldtrichloroacetic-acid insoluble product has been studied. Almost all the four nucleotidedependent incorporating activity is localized in the IOOO× g fraction of such extracts. The RNA character of the product is indicated by the dependence of the reaction on all four nucleotides (ATP, GTP, UTP, and CTP), by the fact that the product is readily solubilized by RNAase treatment, and the fact that both ATP and GTP are incorporated at comparable rates. The incorporation of [3zP]ATP is enhanced by the addition of an ATP-generating system. The system is inhibited by the presence of DNAase, RNAase, or actinomycin D in the incubation mixtures. The product of incorporation is solubilized by RNAase but not by DNAase. Pretreatment of the IOOO x g fraction with DNAase or actinomycin D destroys the RNA synthesizing capacity. Within the IOOO×g fraction, RNA synthesizing activity was associated with both chloroplasts and nuclei, the former being the major site of activity.

INTRODUCTION

A number of reports have been made recently on the characteristics and localization of cell-free systems from higher plants which are capable of synthesizing an RNAlike product. In general, the incorporation of one labeled nucleoside triphosphate has been shown to be enhanced by the presence of the other three nucleoside triphosphates. The RNA nature of the product has been inferred from this fact, together with such properties of the product as RNAase susceptibility, insolubility in trichloroacetic acid and perchloric acid, and solubility in NaC1 solutions. Such systems have been isolated from young pea seedlings1, * and tobacco leaves 3-5. The major site of RNA synthesis in pea seedlings is believed to be the chromatin of the nuclear fraction 6. Similarly the nuclear fraction of tobacco leaves appeared to contain the maior portion of the RNA-synthesizing activity 3. However, in none of this work has the product been shown unequivocally to contain all four ribonucleotides linked by internucleotide bonds. * Permanent address: Institut Agronomique de l'Etat, Gembloux (Belgium). ** Permanent address: Division of Plant Industry, C.S.I.R.O., Canberra (Australia). Abbreviation: PEP, phosphoenolpyruvic acid.

Biochim. Biophys. Acta, 91 (I964) 2o5-216

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2o6

This paper will describe a DNA-dependent system from tobacco leaves which will synthesize R N A when provided with ATP, CTP, UTP, and GTP. Particular emphasis has been placed on the characterization of the synthesized product as RNA. Evidence of the formation of internucleotide bonds in the synthesized product will be presented in the companion paperL MATERIALS AND METHODS

Preparation o/ the lea/ extract Plants of Nicoliana glutinosa and Nicotiana tabacum were grown in a greenhouse and leaves were harvested in the morning. In order to minimize as much as possible disruption of nuclei and chloroplasts, the method employed by SPENCER AND WILDI~ANs for preparing cell-free extracts capable of protein synthesis was also used for the preparation of these extracts. The middle and large veins were removed from the leaves and discarded. Aliquots of 5 g fresh weight of leaf laminae were cooled and chopped to a fine mince with razor blades in 5-6 ml of extracting medium (referred to as "sucrose-Tris" medium) consisting of 0.4 M sucrose, 0.05 M Tris buffer (pH 7.8), 6. 9 m ~ magnesium acetate, lO.3 mM KC1 and 3-9 mM mercaptoethanol. The resulting extract was filtered through 3 layers of fine cloth to remove intact cells and the filtrate was centrifuged for 5 - I o m i n at i o o o × g . In most experiments the supernatant fraction was discarded and the pellet resuspended in 2.5 ml of sucroseTris medium. This suspension will be referred to as the IOOO×g fraction. Low temperature was maintained throughout these procedures. Microscopical examination using previously described techniques 8 showed this material to consist in the main of intact chloroplasts and nuclei together with starch grains and crystalline material. In some experiments further separation of chloroplasts and nuclei was achieved by additional steps of low-speed centrifugation. The "chloroplast" fraction was obtained as follows: the iooo ×g suspension was spun for 2 rain at IOOO×g to produce a stratified pellet, the green upper layer was carefully resuspended in sucroseTris medium without disturbing the lower white layer, and then spun again at IOOO× g for 5 rain. The supernatant was discarded and the green upper layer of this pellet resuspended as before in sucrose-Tris. The same process was repeated 2-3 times more, to produce a final suspension of chloroplasts virtually free of intact nuclei and nearly free of small particulate materials. The "nuclei" fraction was obtained b y spinning the IOOO×g suspension for 2 min at IOOO×g, discarding the supernatant and the green upper part of the pellet, and resuspending the white bottom layer of the pellet in sucrose-Tris medium. The process was repeated several times to yield a suspension rich in intact nuclei and containing relatively few chloroplasts. This fraction was heavily contaminated with starch grains and crystals.

Standard assay /or nucleotide incorporation The reaction mixture consisted of o. 4 ml of leaf extract, phosphoenolpyruvate (580 #g), and pyruvate kinase (Io #g), a mixture of 3 nucleoside triphosphates, and either ES-14C]ATP (or ES-14C]GTP) or E~-a2P;ATP (or ~-32P]GTP). With radioactive ATP, the added nucleoside triphosphates were CTP, GTP and UTP, whereas with radioactive GTP these were ATP, CTP and UTP. The amount of radioactiw~ nucleotide was adjusted to have 5-1o a counts/rain present in each assay, and the Bioctzim. Biophys. Acta, 91 (1964) 2o5-216

RNA SYNTHESIS BY TOBACCO LEAVES

207

isotopic material was added immediately before the incubation which was carried out at 25 °. The concentration of the three other nucleotides was Io-fold higher than the labeled GTP or ATP, since it was determined by experiment that a ratio of I ATP : IO of each of the other 3 nucleosides induced a near m a x i m u m incorporation of ATP. The total volume of the assay mixture was 0.5 ml. After incubation, the tubes were rapidly cooled in ice, and 0.2 ml ATP (or GTP) (12.5 mg/ml) was added, followed b y cold trichloroacetic acid to give a final concentration of 5 %. Using a previously described s method for washing precipitates, the trichloroacetic acid insoluble fraction was sedimented for IO rain at 12 ooo × g and the clear supernatant discarded. The tubes containing the pellets were rapidly frozen in solid CO 2 for 15 min, thawed and resuspended in 5 % cold trichloroacetic acid, transferred quantitatively to Millipore filters (pore size 1.2 ~) and washed 4-5 times with 5-ml aliquots of 5 % trichloroacetic acid. The filters were cemented to planchets, dried and counted with a Nuclear Chicago gas flow counter fitted with a thin Micromil window. Values presented in the tables are the mean of duplicate assays; in most cases, the difference between the mean and the actual values was less than IO %. Activities are expressed in counts/rain incorporated per assay after correction for zero time control values. For the IOOO×g fractions, the zero time controls obtained by adding the radioactive ATP (or GTP) at the end of the incubation period varied from 17-5o counts/rain with a mean from m a n y determinations of 25.

Reagents P y r u v a t e kinase, mercaptoethanol, CTP and U T P were obtained from the California Corporation for Biochemical Research and the tricyclohexylamine salt of phosphoenolpyruvic acid from Boehringer and Soehne, Mannheim. Crystalline pancreatic RNAase and DNAase together with G T P were purchased from Sigma Chemical Company; A T P from Pabst Laboratories; actinomycin D and chloramphenicol from Merck and Co., Inc.; [8-14C~ATP (lO7 counts/min//zmole) and[14C~GTP from Schwarz BioResearch, Inc. [~-s~P]ATP and [~-32P]GTP were synthesized and characterized as described in the companion paper ~. The activities of the several preparations ranged from o.8-4.5-1o ~ counts/min/#mole. Chlorophyll was assayed b y the method of ARNON9 and I)NA by the method of B U R T O N lo.

EXPERIMENTAL RESULTS

Nucleotide dependence The IOOO× g fraction of cell-free extracts of N. glutinosa and N. tabacum leaves catalyzes the incorporation of both laC-and 32P-labeled A T P and GTP into a cold trichloroacetic acid-insoluble product. In all cases the m a x i m u m level of incorporation was dependent on the presence of three other nucleoside triphosphates: UTP, CTP, and either G T P or A T P (Table I). Where conditions were directly comparable (Table I, Expt. I) the incorporation of A T P and GTP was found to proceed at approximately the same rate. The incorporation of A T P is dependent On the presence of all four nucleoside triphosphates for m a x i m u m activity, and no stimulation was observed upon the addition of CTP and GTP alone, or of U T P and GTP (Table II). Biochim. Biophys. Acta, 9I (1964) 2o5-216

j. SEMAL et al.

208

TABLE I INCORPORATION

OF

RADIOACTIVE

ATP

N. tabacum N. tabacum

GTP

BY

THE

IOOO X g

FRACTION

OF

LEAVES

Nuc l e ot i de s added

Incorporation (counts]rain~assay)

[~4C]ATP* ~a~C]ATP + CTP + G T P + U T P I14C]GTP ** [~aC]GTP+ A T P + U T P + CTP [3~p]ATP*** [ 3 2 P ] A T I ' + G T P + C T P + UTP [32p] GTP*** E32p]GTP + ATP + CTP + U T P

66 194 2 48 72 653 53 629

Plant

N. glutinosa

AND

* Specific activity, lO ~ counts/min//zmole. *" Specific activity, 3.5" IO5 counts/min//zmole. *** Specific activity, 4.5" lO7 c o u n t s / m i n / # m o l e . T A B L E I1 INFLUENCE OF CTP, G T P AND U T P ON THE INCORPORATION OF L32P]ATP BY THE IOOO× g VRACTION

OF

TOBACCO

LEAVES

I n c u b a t i o n time, 3 h for E x p t . I and i h for E x p t . 2. D N A a s e concentration,

2 #g/ml

I n c o r p o r a t i o n (counts~rain~assay) Nucleotides added

[32P]ATP [3~P]ATP + [3~p]ATP+ [a2p]ATP+ [asP]ATP+

Expt. t

CTP GTP GTP + UTP CTP + G T P + U T P

Expt. 2

No D N A ase

+ D N A ase

No I ) N A ase

+ D N A ase

423

397

31 23

30 12

437 342 995

4 °2 296 393

421

18

It was furthermore found lhat incorporation induced by all four nucleoside triphosphates was abolished by the presence of DNAase in the incubation mixtures whereas the incorporation of ATP alone was not affected. The presence of an ATP-generating system (pyruvate kinase and PEP) enhances the incorporation of [~-3~P]ATP more than twofold (Table I l i ) , indicating that the active moiety of the incorporation reaction is the nucleoside triphosphate, T A B L E 1II EFFECT

OF

AN

ATP-GENERATING BY

THE

IOOO x g

SYSTEM FRACTION

ON OF

THE

INCORPORATION

TOBACCO

OF

[32P]ATP

LEAVES

I n c u b a t i o n time, 3 h. Incorporation (counts/rain/assay) Nucleotides

added

[82P]AT P [a2p]ATP+CTP+ GTP+ UTP

A TP generating system* Present

A bsent

425 1425

I66 595

* 1.25 #moles of p h o s p h o e n o l p y r u v a t e and io/~g of p y r u v a t e kinase. Biochim. 2Biophys..4eta, 91 (1964) 2o 5- 216

RNA SYNTHESIS BY TOBACCO LEAVES

2o9

rather than the di- or mono-form. This is true both for the incorporation of [32P]ATP alone and in the presence of GTP, UTP, and CTP.

Time dependence In early experiments with the IOOOx g fraction from N. glutinosa, the incorporation of [14C]ATP in the presence of CTP, GTP, and U T P started without a lag and remained linear for 3 h. With the standard IOOO×g flaction from N. tabacum leaves, incorporation was linear for about I h as shown b y the data of Fig. I. In-

"Chloroplasts" 1000

u~

80o

4.

-~ 600

o o

4oo

/

o

+CTP+

P

o

- 200 "Chloroplasts'''

0

3o

;o

Time(rnin)

' 180

Fig. I. Time dependence of incorporation of radioactive ATP and GTP by tobacco leaf extracts. The IOOO× g fraction and "chloroplasts", prepared as described in METHODS, represent separate experiments.

corporation into this fraction declined with further incubation. This decline was not observed with a washed "chloroplasts" preparation, suggesting that RNAase m a y be removed b y washing, thus preventing destruction of the product of incorporation.

E[/ect o[ inhibitors A comparison of the action of DNAase, RNAase and actinomycin D permits some conclusions to be drawn concerning the nature of the incorporation reaction and of the product of this reaction (Table IV). The incorporation of both [~-z2P]ATP and GTP in the presence of three other nucleoside triphosphates was found to be inhibited by either DNAase, RNAase, or actinomycin D. Chloramphenicol was without effect. In contrast, the low endogenous level of incorporation, in the presence of labeled ATP or G T P alone, was much less sensitive to these inhibitors. Furthermore, as seen previously in Table II, this increased sensitivity to DNAase was not found upon addition of one, two, or three nucleotides, but was dependent on the presence of all four nucleoside triphosphates in the reaction mixture. Biochim. Biophys. Acta, 91 (1964) 2o5-216

21o

j.

SEMAL et al.

T A B L E IV INCORPORATION OF [32p]ATP AND [a2P]GTP BY THE IOOO×g FRACTION OF TOBACCO

LEAVES

IN

THE

PRESENCE AND

OF

RNAAsE,

ACTINOMYCIN

DNAAsE,

CHLORAMPHENICOL

D

incubation time, 3 h for E x p t s . i and 2; i h for E x p t s . 3, 4, and .5. Incorporation (counts/rain~assay)

Expt.

Nucleotidcs

Inhibitor

;a2p]AT P

I

None RNAase, I / * g / m l DNAase, I # g / m l Actinomycin D, IO/*g/ml

2

None

la2t']ATI'+ CTP +GTP+UTP

;aet,]GT1 ~ "

/a2PJGTt'+ATI -t C T P + U T P

~

727 153 231 212

293 214 257 331

437 28 396

Actinomycin D, IO/,g/nil Chloramphenicol, 200/,g/nil 3

None DNAase, lO/*g/ml

185 93

816 89

94 33

668 65

4

None RNAase, IO/*g/ml

96 28

456 25

46 34

528 9

5

None

280 168

8oo lOO

Actinomycin D, i o / , g / m l

The effect of arange ofconcentrationsofDNAase and RNAase on bothendcgenous incorporation and incorporation of [a2P]ATP in the presence of CTP, UTP, and GTP is shown in Table V. In the presence of all four nucleotides, o.I/~g of DNAase or RNAase/ml caused 65 and 75 % inhibition, respectively, and in both cases inhibition was almost maximal at I/,g/ml. The greater insensitivity of the incorporation of [32p]ATP alone to these inhibitors is also confirmed here; DNAase and RNAase at IOO #g/ml caused 34 and 64 % inhibition, respectively. TABLE V INCORPORATION

OF

[a=p]ATP BY

INCREASING

THE

AMOUNTS

IOOOXg OF

FRACTION

RNAAsE

AND

IN

TItE

PRESENCE

OF

DNAAsE

Incubation tinle, 3 h. Incorporation (counts/min/assay)

Expt. I

Inhibitor

Concentralion (Hg/ml)

RNAase

Nil

la2pTA T P alone

[a~-t)/A T P + C T P + UTP + GTP

318

1589 39 ° 187 146 lO4

O.I I iO

Expt. 2

DNAase

IOO

1 I8

Nil

389

O.I 1

IO

ioo

257

3o56 Io51 433 398 347

Biochim. Biophys. A~ta, 91 (1964) 2o5-216

RNA

SYNTHESIS BY TOBACCO LEAVES

211

By means of pre-incubation and post-incubation treatments, an effort was made to test whether these inhibitors acted on a component of the reaction or on the product of the reaction. Following a 3-h incubation, a standard reaction mixture containing all four nucleotides was treated for one minute with either IOO #g of DNAase or RNAase/ml. As shown by the data in Table VI, the product of the reaction was TABLE VI EFFECT OF RNAAsE AND DNAASE [ n 2 P ] A T P B Y T H E I 0 0 0 )< g F R A C T I O N

ON THE PRODUCT OF INCORPORATION OF TOBACCO LEAVES IN THE PRESENCE

OF OF

U T P , G T P AND C T P

Incubation time, 3 h. Concentration of DNAase and RNAase, ioo # g / m l . Inhibitor present

During

Mixed ]or x rain a#er incubation

incubation

Expt.

Incorporation (counts/minlassay)

i

None None RNAase

None RNAase None

1589 188 lO 4

Expt. 2

None None None RNAase DNAase

None RNAase DNAase None None

309 ° 264 i 613 I 15 347

found to be far more sensitive to RNAase than to DNAase. Post-incubation with RNAase solubilized 9 ° % of the incorporated [3*P]ATP, whereas similar treatment with DNAase reduced incorporation by 48 %. The effect of RNAase and DNAase present during incubation is also given as a measure of the maximum inhibition to be expected under the same conditions. In contrast, incorporation of [z2P~ATP in the presence of the other three nucleotides was far more sensitive to a pretreatment with either DNAase or actinomycin D than pretreatment with RNAase. Table VII shows the data obtained from an TABLE VII EFFECT OF PRETREATMENT OF THE IOOO X g FRACTION OF TOBACCO LEAVES WITH RNAAsE, DNAASE AND ACTINOMYCIN D ON THE SUBSEQUENT INCORPORATION OF [32P]ATP IN THE PRESENCE OF CTP + GTP + UTP

Concentration of R N A a s e , i / ~ g / m l ; A c t i n o m y c i n D, i o / ~ g / m l . Pretreatment ]or x h with inhibitor ]ollowed by washing and incubation

Expt. I

None RNAase None DNAase None None

None None RNAase None DNAase None Actinomycin D

None RNAase None

None None RNAase

Actinomycin D Expt. 2

Inhibitor present in the mixture during incubation

Incorporation (counts/rain/assay)

1675 607 46 20 12 7 lO8 48 305 199 33

Biochim. Bi ophy s . Acta, 91 (1964) 2o5-216

j. SEMAL et al.

212

experiment in which the iooo ×g fraction of tobacco leaves was preincubated for I h at 25 ° with either RNAase, DNAase or actinomycin D. After pre-incubation the iooo x g fraction was washed three times by centrifugation and resuspended in fresh sucrose-Tris medium in order to remove as much of the inhibitors as possible. The washed, pre-incubated i o o o × g fraction was then incubated as usual with [a~p3ATI? , CTP, U T P and GTP. Control extracts were preincubated in the absence of inhibitors, then washed and assayed both in the presence and absence of inhibitors. Pretreatment with DNAase and actinomycin D reduced incorporation by 99 and 94 % respectively, whereas lRNAase pretreatment caused only 35 °o inhibition in one experiment and 64 % in another. When 1RNAase was added directly to the reaction mixture it was more inhibitory than DNAase. From the above inhibitor studies it can be concluded that the four nucleotidedependent incorporating system contains an essential DNA component, and that the product of the reaction is RNA-like in character.

Localization o/the incorporating activity in cell-/ree hornogenates A tobacco leaf homogenate was fractionated by differential centrifugation and the following fractions were tested for their ability to incorporate [s2P~ATP both alone and in the presence of GTP, CTP, and UTP: (a) the IOOO×g pellet obtained after centrifugation of the leaf homogenate for 5 rain at IOOO×g; (b) the IOOO×g supernatant, so obtained; (c) the 144 ooo ×g pellet obtained after centrifuging the IOOO x g supernatant for 9 ° min at 144 ooo xg; (d) the 144 ooo ×g supernatant, so obtained. Each pellet was resuspended in its original volume of fresh sucrose-Tris medium. The data in Table V I I I show that 95 o~ of the four nucleotide-dependent incorporating activity resides in the IOOO×g fraction. The other fractions are less active and show little, if any, stimulation b y the presence of the other three nucleoside triphosphates. TABLE INCORPORATION

OF

Ea2PjATP I N T O

All p e l l e t s w e r e r e s u s p e n d e d

VIII

DIFFERENT

]tRACTIONS

in their original volume

OF

TOBACCO

LEAVES

of f r e s h m e d i u m .

Incorporation (counts/min/assa3,) Fraction

IOOO X g p e l l e t IOOO × g s u p e r n a t a n t 144 o o o x g p e l l e t 144 o o o X g s u p e r n a t a n t

[s21~TAT P

[ s:PJA T P + CT I'

16o iio 41 3°

229 ° i24 3° 25

+GTP

+UTI )

The incorporating activity of the IOOOx g fraction is firmly associated with a particulate, readily sedimented component. This is shown by the fact that such fractions can be subjected to four successive washings by centrifugation at IO ooo × g and resuspension in fresh sucrose-Tris medium without appreciable loss in activity (Table IX). Although some loss of activity occurred with the first washes this was largely due to a failure to recover all tbe particulate components by the centrifugation step. This is evidenced by the fact that when incorporating activity is expressed on a Biochim.

B i o p h y s . A c t a , 91

(1964) 205 216

RNA

SYNTHESIS BY TOBACCO LEAVES

213

TABLE IX EFFECT OF WASHING THE IOOOX g FRACTION ON SUBSEQUENT INCORPORATION OF [s2P]GTP IN THE PRESENCE OF A T P + C T P + U T P Incubation time, 3 h

Incorporation Number o/washes

None I 2 3 4

Counts/rain~assay

Counts/rain~rag chlorophyll

3o84 2o12 1816 1857 1844

47 38 45 46 45

5° o 2oo 15o 6oo 59o

chlorophyll basis, it was found to be relatively constant throughout the four washes. However, no direct correlation of activity with the chloroplasts alone can be inferred, since other organelles, including nuclei, would also be sedimented under these washing conditions. In other experiments, the activity increased with the first two or three washes, presumably due to the removal of an endogenous inhibitor such as RNAase.

Localization o/ the incorporating activity within the IOOO×g [faction Since the IOOO×g fraction is a mixture of intact and fragmented nuclei and chloroplasts together with a small proportion of other microscopically visible, non-green particulate materials, an attempt was made to localize the incorporating activity in relation to these structures. After incubation of the IOOO×g fraction with [~P~ATP, GTP, CTP, and U T P the incubated reaction mixture was placed on a Ficoll density gradient to separate the intact nuclei and nuclear fragments from the chloroplasts as described elsewhere s. The nuclei were recovered and the amount of incorporation into this fraction was determined. In a second experiment, after incubation, the reaction mixture was centrifuged for I h at IOO ooo ×g to produce a supernatant solution that would be expected to contain any counts associated with soluble RNA or materials otherwise solubilized or separated from the chloroplasts and nuclei. As shown by the results in Table X, only 5 % of the incorporated counts were associated with the nuclei, and only 15 % were present in the supernatant after TABLE FRACTIONATION

X

OF TOBACCO I O O O × g FRACTION AFTER INCUBATION W I T H [s2P]ATP+ CTP+ GTP+ UTP

T i m e of incubation, 3 h for Expt. x and 2.5 h for Expt. 2. T h e nuclei were separated fromchlorop l a s t s as a p e l l e t b y m e a n s of a Ficoll d e n s i t y g r a d i e n t . S u p e r n a t a n t a n d p e l l e t o b t a i n e d a f t e r c e n t r i f u g a t i o n of t h e i o o o × g p r e p a r a t i o n for I h a t 39 ooo r e v . / m i n i n Spinco c e n t r i f u g e w i t h r o t o r S W 39.

Expt.

Fraction

Incorporation

I

IOOO × g Nuclei

716 33

2

IOOO X g Supernatant Pellet

386 56 255

Biochim. Biophys. Acta, 91 (1964) 2o5-216

2i 4

j. SEMAL et al.

high-speed centrifugation. These results suggest that the major part of the incorporated radioactivity is associated with material other than the nuclei and s-RNA. Further evidence of the relatively minor contribution of the nuclei to the total incorporation by the IOOO×g fraction was also found in experiments where some separation of chloroplasts and nuclei was made before incubation. For this purpose, enriched "chloroplast" and "nuclei" fractions were prepared as described in METHODS. Although the separation into chloroplasts and nuclei was far from complete, their distribution was greatly altered relative to that in the I o o o × g fraction. Equal volumes of both "chloroplast" and "nuclei" preparations (each originating from the same weight of leaf material) were incubated either separately or in combination, and the capacity for incorporation of [a2p]ATP in the presence and absence of the other three nucleotides was determined. The concentration of each separate fraction was adjusted with sucrosc-Tris medium to be the same as in the combined preparation. It was found (Table XI) that the four-nucleotide-dependent incorporation had been TABLE INCORPORATION OF

XI

[a2P]ATP INTO " C H L O R O P L A S T S " AND " N U C L E I " FRACTIONS FRO3,I TOBACCO LEAVES

I n c u b a t i o n t i m e , 3 h for E x p t . I a n d i h for E x p t . 2. C h l o r o p l a s t s a n d n u c l e i p r e p a r e d as d e s c r i b e d in

METHODS.

Chlorophyll content "chloroplasts" Chlorophyll content "nuclei" ~ 4o;

D N A c o n t e n t " c h l o r o p l a s t s " = o.S. DNA content "nuclei"

Incorporation (counts[rain/assay. [a~Pj,4TP alone

Expt. I

Expt. 2

i volume "chloroplasts" + ~ volume "sucrose-Tris" i volume "nuclei" -i- i v o l u m e " s u c r o s e T r i s " I volume "chloroplasts" + r volume "nuclei" I volume "chloroplasts"

/:JzP],4TI~+CTP + G T P -~- U l" P

4

637

lO5

276

19o

I200

37

634

23

81

66

705

-1- I v o l u m e " s u c r o s e T r i s "

I volume "nuclei" + I volume "sucrose-Tris" volume "chloroplasts" "1- 1: v o l u m e " n u c l e i "

concentrated in the "chloroplast" fraction. Both the degree of stimulation of [a2p]_ AT]? incorporation b y UTP, GTP, and CTP, and the absolute level of incorporation with four nucleotides was much higher in the "chloroplast" than in the "nuclei" fraction. However, the level of incorporation by these two fractions could not be directly correlated with their relative contents of chlorophyll or DNA, since the "chloroplast" fraction contained forty times as much chlorophyll and o.77 as much DNA as the "nuclei" preparation. Thus, although the evidence points to the "chloroplast" fraction as the major site of RNA synthesis, there is no clear evidence which implicates the chloroplasts themselves rather than some contaminating component of this fraction as the precise site of the synthetic system. Biochim. Biophys. iqcta, 91 (i964) 205 216

RNA

SYNTHESIS BY TOBACCO LEAVES

215

DISCUSSION

All the evidence from the above experiments suggests that tile cold trichloroacetic acid insoluble product of the nucleotide incorporating system in tile IOOOx g fraction from tobacco leaves is indeed RNA. First, the reaction is strongly dependent on the presence of all four nucleoside triphosphates for maximum activity (Tables I and II). Second, labeled ATP and labeled GTP are incorporated at approximately the same rate in the presence of the remaining three nucleotides (Table I). The incorporation of only ATP might be interpreted as the formation of polyadenylic acid, but the comparable incorporation of GTP makes this interpretation unlikely in view of the apparent non-existence of a polyguanylic acid synthesizing system in nature. Again, the large stimulation in E32P]ATP incorporation brought by the addition of an ATP-generating system (Table III) indicates that tile triphosphate moiety is the true substrate, and argues against the participation of nucleoside diphosphate phosphorylase. The fact that E32P]ATP incorporation is not enhanced by CTP alone (Table II) suggests that the repair of the terminal CpCpA sequence of partially degraded soluble RNA does not make a significant contribution to the total incorporation. Finally, the rapid solubilization of tile product of E~2P]ATP incorporation by brief RNAase treatment (Table VI) is again indicative of the RNA-like nature of the product. All the above evidence concerning the nature of tile product is indirect and correlative. Direct proof that the product is indeed RNA is provided in the companion paper ~in which the labeled product is shown to contain all four nucleotides linked by internucleotide bonds. The sensitivity of the incorporating system of the IOOOXg fraction to preincubation with DNAase and actinomycin D (Table IV) is indicative of an essential DNA component directing the RNA synthesis. The system from tobacco leaves thus resembles most RNA synthesizing systems described for normal animal and bacterial cells11,1~ and is in accord with the findings of SANGER AND KNIGHTla who recently found that the incorporation of [2-14C]uracil into the RNA of intact tobacco leaves is also inhibited by actinomycin D. Both tile DNA and the associated polymerase enzyme must be firmly associated with particulate components of the IOOO×g fraction, since repeated washings of this fraction failed to lower its incorporating capacity (Table IX). In unpublished experiments, it was found that the addition of exogenous tobacco DNA did not enhance the activity of the washed IOOOx g fraction. This was also the case when washing was preceded by DNAase treatment. Microscopic examination of cell-free extracts obtained by our procedures showed a good degree of preservation of tile structural integrity of tile various cell components. Under these conditions 95 % of the RNA synthesizing activity of the extracts was localized in the IOOO×g pellet (Table VIII) which contains chloroplasts, nuclei, and some microscopically visible, smaller, non-green particles of the size of mitochondria and spherosomes. The residual activity found in the IOOO×g supernatant fraction was not stimulated by tile presence of all four nucleotides and is presumably due to an ATP polymerase (Table Vllr). It is possible that this latter system is the same as that which KARASEKAND SCHRAMM4 isolated from the IOO ooo × g supernatant fraction of an extract of tobacco leaves. Although it is clear that tile components of the IOOO×g fraction comprise tile only significant RNA synthesizing system of the leaf cell, the precise localization of Biochim. Biophys. Acta, 91 (1964) 2o5-216

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the system w i t h i n the IOOO× g fraction is less clear cut. I n terms of i n c o r p o r a t e d counts only 5 % were associated with the nuclei after i n c u b a t i o n (Table X). F u r t h e r more, prior separation of the IOOO×g fraction into a chloroplast-enriched a n d a nuclei-enriched fraction before i n c u b a t i o n showed t h a t a m a j o r p o r t i o n of the a c t i v i t y was associated with the chloroplast fraction (Table XI). However, no simple correlation was found b e t w e e n the i n c o r p o r a t i n g a c t i v i t y a n d the ratio of the chlorophyll c o n t e n t of the two fractions. Our conclusion is t h a t b o t h chloroplasts a n d nuclei c o n t r i b u t e to the R N A synthesizing activity, with the chloroplasts m a k i n g the m a j o r c o n t r i b u t i o n . T a k e n together with recent evidence of a u n i q u e chloroplast D N A fraction ~4, this i m m e d i a t e l y suggests the possibility t h a t it is the chloroplast D N A which is, to a large measure, directing R N A synthesis. Both the fact t h a t the incorporating system w i t h s t a n d s extensive washing, a n d the fact t h a t the purified chloroplast p r e p a r a t i o n s are more active t h a n the corresponding nuclei preparations, argue against the possibility the R N A synthesis b y chloroplast fraction is being p r i m e d b y c o n t a m i n a t i n g n u c l e a r DNA. Since completion of these experiments, KIRK15 has presented evidence of a D N A a n d four nucleotide d e p e n d e n t , [t4C]ATPi n c o r p o r a t i n g system localized in the chloroplast fraction of broad b e a n leaf extracts. ACKNOWLEDGEMENTS This work was supported in part b y research grants from the Division of Biology a n d Medicine, U. S. Atomic E n e r g y Commission, Contract A T ( I I - I ) - 3 4 , Project 8; the N a t i o n a l I n s t i t u t e s of H e a l t h (AI-00536); the N a t i o n a l Science F o u n d a t i o n (B-I4729). One of us (J. S.) is i n d e b t e d to the Belgian G o v e r n m e n t for a N A T 0 g r a n t t h a t enabled him to visit the U.S. REFERENCES 1 R. HUANG,N. MAHESHWARIAND J. BONNER, Biochem. Biophys. Res. Commun., 3 (196°) 689J. BONNER, R. HUANG AND N. MAHESHWARI, Proc. Natl. Acad. Sci. U.S., 47 (I96I) 1548. 3 R. BANDURSKIAND S. C. MAHESHWARI, Plant Physiol., 37 (1962) 556. 4 ]y[. t{ARASEKAND G. SCHRAMM,Biochem. Biophys. Res. Commun., 9 (1962) 63. s V¢. HUDSON, Y. T. KIM, R. SMITH AND S. \VILDMAN, Bioehim. Biophys. 3cta, 76 (t963) 257. 6 j. RHO AND J. BONNER, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1611. R. MOYER, R. SMITH, J. SEMALAND Y. T. KIM, Bioehim. Biophys. Acta, 91 (1964) 217. 8 D. SPENCER ANn S. WILDMAN, Biochemistry, 3 (1964) 954. 9 D. ARNON, Plant Physiol., 24 (1949) I. 1o K. BURTON, Biochem J., 62 (1956) 315 • 11 S. V~TEISS, Federation Proe., 21 (1962) 12o. 1~ j. FURTH, J. HURWlTZAND M. ANDERS, J. Biol. Chem., 237 (1962) 2611. 1~ H. SANGERAND C. t(NIGHT, Biochem. Biophys. Res. Commun., 13 (1963) 45514 E. CHUN, M. VAUGHANAND A. RICH, J. Mol. Biol., 7 (1963) 13o. is j. KIRK, Biochem. Biophys. Res. Commun., 14 (1964) 393. Biochim. Biophys. Acta, 91 (1964) 205 216