Eur. J. Biochem. 77, 393-400 (1977)

Initiation of RNA Synthesis in Isolated Nuclei Eli GILBOA, Hermona SOREQ, and Haim AVIV Departments of Virology and Neurobiology, Weizmann Institute of Science, Rehovot (Received February 14, 1977)

Ribonucleotide triphosphates, labeled at the 8 position, were synthesized and used directly to quantify RNA chain initiation in nuclei isolated from Friend cells grown in tissue culture. At the optimal salt concentration, low-molecular-weight RNAs (4 - 5 S) synthesized by RNA polymerase I11 were the predominant species initiated. Less than 5 of the molecules were initiated by polymerase 11. We calculate that 50 - 80 % of the small RNA molecules synthesized in vitro were also initiated in vitro. Assuming that a substantial fraction of the nuclei were active in vitro, the number of 4- 5 S RNA molecules initiated per nucleus was about 100 molecules/min. Three classes of eukaryotic RNA polymerases have now been identified, transcribing three types of genes; the genes for ribosomal RNA, for heterogenous nuclear RNA and for small RNA (tRNA and ribosomal 5-S RNA) [l]. Control elements affecting the regulation of transcription of the type described in prokaryotes (initiation and termination factors, repressors and inducers) [2] are not yet known in eukaryotes. The mode by which the eukaryotic genome is regulated is not well understood. The dimensions of the problem may be illustrated by the enormous complexity of the genome and by the fact that only a small fraction of the genome is expressed at any time. Several models have been proposed to explain the organization and expression of the genome [3,4]. Genetic analysis in conjunction with physiological and biochemical studies have been extremely valuable in the elucidation of control elements in prokaryotic systems, and their final identification was facilitated by direct isolation of these factors in cell-free transcription systems [2]. However, several attempts to use purified nucleoproteins (such as chromatin structure) have not been satisfying; in most cases the regulated expression was lost or impaired in vitro [5,6]. In contrast to that, regulating elements in intact nuclei appear to be generally maintained [7,8]. This property, combined with the permeability of nuclei to constituents of low molecular weight, made the cell-free Enzymes. Polynucleotide phosphorylase (EC 2.7.7.8); hexokinase (EC 2.7.1.1): pyruvate kinase (EC 2.7.1.40); creatine kinase (EC 2.7.3.2); bacterial alkaline phosphatase (EC 3.1.3.1): DNAase (EC 3.1.4.5); nuclease endonuclease or nuclease P’ (EC 3.1.4.9); nucleotide pyrophosphatase (EC 3.6.1.9).

systems using isolated nuclei an attractive model for studies on transcription. By analogy with prokaryotic cells, the initiation of RNA should be the major site for regulation of gene transcription. Initiation of RNA synthesis in isolated nuclei was demonstrated by the incorporation of y-labeled nucleoside triphosphates into RNA [9]. However, in these studies the quantification of initiation could not be carefully measured. Indeed, some researchers point out specifically that they have failed to quantify initiation of RNA in vitro using isolated nuclei [lo]. We believe that the use of triphosphates labeled at the 8 position in these studies may help to solve the problems encountered for the following reasons. First, the y-phosphate at the 5’ end of RNA chains may be subjected to phosphorolysis; second, the yphosphate can also be removed during the process of ‘capping’ [ll]. In addition, the y-phosphate may also serve as a donor to phosphorylate proteins, DNA or RNA [12]. These problems should be less severe or absent when 8-labeled nucleotides are used. We therefore developed a procedure for the preparation of 8-labeled ribonucleoside triphosphates which we used to measure initiation of RNA chains quantitatively in vitro in isolated nuclei. The procedure was based on a pre-existing one, developed by Littauer et al. [13]. Reichard et al. have previously prepared and used b-32P-labeled ribonucleoside triphosphates in studies of RNA primed DNA initiation [14]. In the experiments described here we found that most of the RNA chains initiated are synthesized by RNA polymerase 111; very few, if any, of the initiated molecules were transcribed by RNA polymerase 11. Furthermore, we have calculated that only about

394

100 genes of tRNA and 5-S ribosomal RNA are transcribed in vitro, while several thousands of them are present in the genome.

Initiation of RNA Synthesis

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EXPERIMENTAL PROCEDURES Materials Nucleoside di and triphosphates as well as phosphoenolpyruvate, creatine phosphate, hexokinase (170 Ujmg), Escherichia coli RNA polymerase (500 U/ mg) and nucleotide pyrophosphatase were purchased from Sigma. Pyruvate kinase and creatine kinase were ' from Boehringer, P1 nuclease from Yamasha Shoyu Co., DNAase (RNAase-free) from Worthington, and poly(ethy1eneimine)-cellulose plates from MachereyNagel. [32P]Orthophosphateand [5, 6-3H]UTP, 50 Ci/ mmol, were obtained from Radiochemical Center (Amersham, England). Synthesis of Nucleosicie (b-32PJDiphosphates and [p-32P J Triphosphates The method consists of two steps. First, the pphosphate moieties of nucleoside diphosphates (NDP) were exchanged with [32P]orthophosphateusing highly purified E. coli polynucleotide phosphorylase (H. Soreq and U. Z. Littauer, in press). Next, the b-32P-labeled nucleoside diphosphates were phosphorylated by pyruvate kinase. The optimal conditions for exchange were different for each nucleoside diphosphate. Under the optimal conditions for the exchange reaction, about 30-40 % of the [32P]orthophosphate was incorporated into the nucleoside [p32P]diphosphate product. Specific activity values of 32P-labeled diphosphates of 50 Ci/mmol or more were obtained. Conditionsf o r the Exchange Reactions Each reaction mixture of 1 ml contained 50 mM Tris-HCI buffer, pH 8.2, 0.04 mM GDP, ADP, CDP or UDP, and 5 mM MgC12, except for the GDP exchange reaction, where 12 mM MgC12 was used. 0.08 mM 32P-labeled sodium dihydrogen phosphate was used for GDP exchange, 0.068 mM for ADP, 0.02 mM for CDP or 0.04 mM for the UDP exchange reaction and 0.17 mg/ml highly purified E. coli polynucleotide phosphorylase (250 Ujmg) was used. The [32P]orthophosphate obtained from Amersham in diluted HCl, was dried by evaporation and dissolved in the reaction mixture without the enzyme, which was added last. 10 mCi was used in each exchange reaction (1 ml). The extent of exchange was followed by the phosphomolybdate test [15]. When equilibrium was reached (1 - 2 h), the reaction mixture was immediately subjected to phosphorylation.

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Fig. 1. Sepurution oj' [I-"zP-labeled nucleoside triphosphates by thinlayer chromatography. Aliquots of 1 pl, containing (2- 10) x lo4 counts/min of nucleoside [fl-32P]triphosphates,prepared and purified as described, were spotted on poly(ethy1eneimine)-cellulose plates, together with 2- 5 pg unlabeled nucleoside triphosphate. The plates were prewashed with water and dried before application. The chromatograms were developed by 1 M KHzP04, pH 4.2, for 2 h at room temperature and dried. The radioactive materials were identified by 2 h exposure to Kodak X-ray film (XRP-5 Omat) and the migration of the unlabeled nucleoside triphosphates was followed by ultraviolet absorption. The different spots contain nucleoside [fl-32P]triphosphatepurified products: (1) ATP; (2) GTP; (3) CTP; (4) UTP; (5) a mixture of all four nucleoside triphosphates synthesized

Conditionsf o r the Phosphorylation Of'(fl-"PJ

NDPs

The reaction mixtures from the exchange step were adjusted to 20 mM KC1, 10 mM MgC12,6 mM phosphoenolpyruvate, and 4 U/ml of pyruvate kinase were then added and the mixtures incubated for 30 min at 37 "C. Purification of Nucleoside (p-32P]TriphosphateJ ((p-32PJNTP) 0.1 vol. of 70 "/, perchloric acid was added and the mixture was incubated at 0 "C for 15 min. The precipitate was collected by centrifugation in a Zentrifuge 3200 (Eppendorf) for 2 min at 10000 x g and discarded. The supernatant was titrated by an equivalent amount of KOH to neutral pH, and the precipitate collected by a similar spin. The precipitate was rewashed with 0.5 vol. of water. Supernatants were combined, diluted 15-20 times (25 ml) with water and applied to a column of DEAE-cellulose (1 ml) previously equilibrated with water. Free phosphate was removed with triethylamine carbonate, pH 7.9, having a conductivity of 1.7 mS (equivalent to 20 mM NaCI). The nucleoside [p-32P]triphosphate was eluted with triethylamine carbonate, pH 7.9, having a conductivity of 8.5 mS (equivalent to 0.1 M NaCI). This fraction, which contained the [p-32P]NTP was evaporated to dryness at room temperature in a rotary evaporator (Evapomix, Buchler), dissolved in water, re-evaporated and redissolved in a small volume of water. If the pH

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E. Gilboa, H. Soreq, and H. Aviv

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Fig. 2. Analysis of the [32P]orthophosphatemoiety in the synthesized nucleoside (32P]tuipliosphute. (A) Treatment with hexokinase. Each reaction mixture of 100 pl contained 50 mM Tris-HCI buffer, pH 7.8; 7 mM MgC12, 200 mM D(+)-glucose, 2 mM unlabeled ATP or GTP; 2 - 10 x lo6 countslmin [fl-3ZP]ATPor [fl-32P]GTP,respectively, and hexokinase, (10 Uiml for ATP and 50 Ujml for GTP treatment). The reaction mixtures were incubated for 10 min at 37 "C. Aliquots of 2-4 p1 were subjected to high-voltage electrophoresis on Whatman 3 MM sheets (25 x 57 cm) in 50 mM sodium citrate, pH 5.2, at 3000 V for 60 min. Unlabeled nucleotides and radioactive materials were detected as described in Fig. 1. (1) [p-32P]ATP;(2) [p-3'P]ATP after treatment with hexokinase; (3) [p-32P]GTP;(4) [fl-3ZP]GTPafter treatment with hexokinase. (B) Treatment with nucleotide pyrophosphatase. Each reaction mixture of 100 pl contained 100 mM Tris-HCI buffer, pH 7.8; 3 mM MgCI2; 25 mM unlabeled ATP or GTP; ( 2 - 10) x lo6 counts/min [p-3ZP]ATPor [p-3ZP]GTPand 10 pl(2.5 units) nucleotide pyrophosphatase. The reaction mixtures were incubated for 60 min at 37 "C. Aliquots of 2-4 p1 were separated by paper electrophoresis and autoradiographed as described in (A). (1) [/j-32P]ATP;(2) [p-3ZP]ATPafter nucleotide pyrophosphatase treatment; ( 3 ) [/I-~'P]GTP;(4) [/(-3ZP]GTP after nucleotide pyrophosphatase treatment; (5) ["P]orthophosphate marker

was still alkaline, another cycle of evaporation was performed. The yield of purification was about 75 The specific activities of the purified [P-32P]NTPs were directly determined by measuring the absorption of the purified products in the ultraviolet region. The whole procedure is rather convenient and can be completed within one day. Millicurie quantities of labeled triphosphates can be obtained. All four ribonucleoside triphosphates synthesized were separated by thin-layer chromatography on poly(ethy1eneimine)-cellulose followed by autoradiography of the developed plates. As shown in Fig. 1, each of the products was pure from other contaminations. An independent proof that the labeled phosphate was in the /3-position was obtained by treatment of 32P-labeled ATP and GTP with hexokinase, which removes specifically the y-phosphate from NTP (Fig. 2A). The products were further identified by treatment with nucleotide pyrophosphatase, which cleaves the pyrophosphate bonds of nucleoside di and triphosphates, while free pyrophosphate will not serve as a substrate (Fig. 2B). Most of the radioactivity migrated much faster than free orthophosphate, identifying it tentatively as pyrophosphate, while a residual amount of 5 % of the radioactivity migrated as free orthophosphate. No radioactivity co-migrated with unlabelled AMP or GMP, thus excluding the possibility that the phosphate label was incorporated into the rx-position.

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Isolation of Nuclei from Erythroleukemic Friend Cells Friend cells were grown as described previously [16]. Nuclei were isolated essentially as described by Marzluff et al. [17] with one modification. The centrifugation of the nuclei through 2 M sucrose was done in a Sorvall centrifuge at 7000 rev./min for 1 h in a swing-out HS-4 rotor. Nuclei were stored in liquid air.

AssuyJor R N A Synthesis in vitro A standard 50-p1 reaction mixture (in 1.5-ml Eppendorf tubes) contained 40 mM 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid/KOH, pH 8.0, 5 mM MgClz, 1 mM MnC12, 2 mM dithiothreitol, 1 mM spermidine, 0.15 M potassium acetate, 6 mM creatine phosphate, 10 units/ml creatine kinase, 0.4 mM CTP, 0.1 mM ['HIUTP (234 counts min-' pmol-') and either 0.4 mM GTP and 50 pM [/3-"P]ATP or 2 mM ATP and 50 pM [/3-32P]GTP(exact specific activities are indicated in each experiment), as well as 1.8 x lo6 nuclei. The reaction mixture was incubated at 25 "C for 60 min. The reaction was stopped by the addition of 5 p10.1 M ATP, 5 pl5 M NaCI, 2 1.11 1 M MgClz and 2 pl DNAase, 2 mg/ml. After 2 - 3 min incubation on ice, 5 p10.5 M EDTA and 2 pl 10 sodium dodecylsulfate were added. Nucleic acids were extracted by the hot phenol method essentially as described by Penman [18]. 70 pl phenol was added, the mixture shaken, heated briefly to 60 "C, reshaken,

396

and 70 pl chloroform was added. The shaking and heating were repeated and the mixture was centrifuged for 1 min at 10000 x g in a Zentrifuge 3200 (Eppendorf). The organic phase was discarded and the aqueous phase plus interphase were extracted twice in a similar manner with 70 p1 chloroform only. The final aqueous phase was removed and the organic phase was re-extracted (without heating) with 100 pl water. The two aqueous phases were pooled and further processed. The RNA synthesized was separated from the labeled triphosphates by the following procedure. A plastic 5-ml disposable syringe was stoppered with glass-wool and filled with swollen Sephadex G-50 in 10 mM Tris, pH 7.5, containing 0.1 M NaCl and 1 mM EDTA. The syringe was spun in a GLC centrifuge (Sorvall) at 3000 rev./min for 5 min and the effluent was discarded. The phenol-extracted reaction mixture was carefully loaded on top of the same Sephadex G-50 column and respun at the same speed for 30 min. To the effluent, 20 pg of salmon-sperm DNA was added and nucleic acids were precipitated with trichloroacetic acid and counted. The background of this procedure was less than 0.0001%. A typical reaction mixture consisted of 20-30 pCi of 32Plabeled NTP. Incorporation into RNA was around 500- 1000 counts/min (0.001 %). Analysis of 12 reaction mixtures was accomplished in about 3 - 4 h. RESULTS Kinetics of R N A Chain Initiation The presumed stability of the P-phosphate as compared to y-phosphate in the RNA chains initiated in vitro, and the convenience of the procedures employed, enabled a systematic search for the optimal conditions for chain initiation. The effect of salt concentration on RNA synthesis was measured by the incorporation of [P-32P]GTP and [P-32P]ATP into RNA. As seen in Fig.3 the optimum for elongation, was rather different from that for initiation. Incorporation of [P-32P]GTP was much higher than the incorporation of [fl-32P]ATPand the salt optimum was rather broad, (0.1 - 0.15 M potassium acetate or the equivalent ionic strength provided by ammonium sulfate, not shown), while the incorporation of [P32P]ATPwas only slightly affected by salt (Fig. 3A, B). The reason for the difference between the shape of the curve when P-labeled ATP or GTP was used is not clear. Does it represent different polymerases or subunits thereof? In contrast to initiation, the incorporation of [3H]UTP had a much higher optimum for salt (Fig. 3C, D). The contribution of the different RNA polymerases was determined by their sensitivity to a-amanitin. While chain elongation (i.e. UTP incorporation) was

Initiation of RNA Synthesis

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remarkably inhibited by a low concentration of aamanitin (2 pg/ml), the incorporation of [P-32P]ATP and [B-32P]GTPwas hardly affected, if at all (Fig.3 and Table 1). It is clear that while 70- 80 % of the incorporation of [3H]UTP was contributed by polymerase I1 and only a small percentage was contributed by polymerase 111, most of the incorporation of plabeled ATP and GTP was due to polymerase 111. The incorporation of [3H]UTP proceeded under these conditions for at least 120 min. The incorporation of P-labeled ATP and GTP is also remarkably long (Fig. 4). Incorporation of fi-32P-labeled ATP and GTP was sensitive to actinomycin D. Incorporation of P-labeled CTP and UTP was remarkably less than 1/10 that of ATP or GTP (not shown).

5 '-End Analysis ojthe R N A Synthesized in vitro That the P-32P-labeledGTP and ATP were indeed incorporated at the 5' end of the RNA chain was shown by the following procedure. The RNA synthesized was exhaustively digested by a mixture of ribonucleases : pancreatic RNAase A, RNAase TI and T2, which should produce for every initiated chain one 32P-labeled ribonucleoside 5'-triphosphate 3'-monophosphate. The digested RNA was subjected to high-voltage electrophoresis. It can be seen in Fig. 5A and B that the 3H-labeled material co-migrated with UMP, while the migration of 32Pwas distributed into two fractions. 35 of the radioactivity migrated

397

E. Gilboa, H. Soreq, and H. Aviv

Table 1. Sensitivity of R N A synthesis to ol-amanitin Reaction mixtures (see Experimental Procedure) were scaled up to 0.5 ml. Time of incubation was 30 min. After extraction with phenol the RNA was precipitated with ethanol, dissolved in 0.2 ml water and passed twice through Sephadex G-50 as described. The proportion of cc-amanitin sensitive incorporation is given in parentheses. (A) [3H]UTP, 234 counts min-' pmol-', and [P-3ZP]ATP,22 100 counts min-' pmol-'. (B) [3H]GTP, 324 counts min-' pmol-', and [p-32P]GTP,14000 counts min-' pmol-'

(B) Incorporation of

(A) Incorporation of

a-Amanitin

0 2 200

[3H]UTP

[D-32P]ATP

['HIGTP

[p-32P]GTP

124 21.5 (82.7) 18.4 (85.2)

0.145 0.140 (3.5) 0.054 (62.8)

194 62.5 (67.8) 48.5 (77.50)

0.17 0.18 0.06 (64.7)

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Fig.4. Kinetics of R N A synfhesis in isolutednuclei. (A) [3H]UTP, 234 counts min-' pmol-', and [D-32P]GTP,11600 counts min-' pmol-'. (B) L'HIUTP, 234 counts min-' pmol-', and [P-32P]ATP,27000 counts min-' pmol-'. For conditions of reaction see Experimental Procedures. [fl-"P]NTP (a); [3H]UTP (0)

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Fig. 5. Analysis o f t h e 5' ends o f R N A synthesized in vitro. Reaction mixtures (sec Experimental Procedures) were scaled up to 0.5 ml. Time of incubation was 30 min. After extraction with phenol the RNA was precipitated with ethanol, dissolved in 0.2 in1 water and passed twice through Sephadex G-50 as described. The purified RNA was digested with RNAase A, 25 pgjml, RNAase TI 25 pglml and RNAase Tz, 10 units/ml in 25 mM ammonium acetate pH 5.0, for 4 h at 37 "C. After incubation the pH was raised to about 7.8 with Tris-HC1 and split into two. To one half PI nuclease was added to a final concentration of 1 mg/ml. The reaction mixtures were further incubated for 60 min at 37 "C, and subjected to high-voltage electrophoresis as described in Fig. 2A. After drying the mobilities of non-labeled nucleotides were determined by ultraviolet absorption. I-cm-wide strips were cut and counted. (A, C) RNA was synthesized in the presence of [3H]UTP(234 counts min-' pmol-') and [P-32P]GTP(19000 counts min-' pmol-'). (A) After treatment with RNAase. (C) After subsequent treatment with PI nuclease. (B, D) RNA was synthesized in the presence of L3H]UTP (234 counts min-' pmol-') and [[j-3ZP]ATP43000 counts min-' pmol-'. (B) After treatment with RNAase. (I)) After subsequent treatment with PI nuclease. 'H ( 0 ) ;"P (0)

398

Initiation of RNA Synthesis

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Fig. 6. Size distribution oynewly synthesized R N A in isolated Friend nuclei. For conditions of RNA synthesis and specific activities of labeled precursors, see Table 1. The purified RNA was dissolved in 10 mM Tris, pH 7.8, and 40 mM MgCL, DNAase was added to 40 pg/ml and the mixture was incubated on ice for 60 min. Reaction was stopped by adding EDTA to 60 mM. Electrophoresis of RNA was on 1.7 ”i, polyacrylamide/0.5 ”,, agarose slab gels according to Peacock and Dingman [19]. Gels were stained with Stains-all (Sigma), sliced to 3-mm slices, each dissolved in 0.3 ml NCS (Eastman) for 60 min at 37 “C, and counted in toluene. (A-C) Incorporation of [3H]UTP and [P-32P]ATP. (D-F) Incorporation of [3H]GTPand [8-32P]GTP.(A, D) Without cc-amanitin. (B, E) 2 pgjml a-amanitin was present. (C, F) 200 pg/pl x-amanitin was present. 3H ( 0 ) ;32P (0)

slightly faster than the corresponding nucleoside triphosphate and the rest migrated substantially faster than its corresponding nucleoside triphosphate. When the RNAase digest was further treated with PI nuclease under conditions where the phosphate bound at the 3‘ position of the ribose moiety of a nucleotide would be removed, all ’H radioactivity migrated as uridine while 32P was distributed into two fractions co-migrating with nucleoside di and triphosphates (Fig. 5 C , D). These data are interpreted as follows. [/h3’P]ATP and [P-32P]GTPwere incorporated exclusively at the 5’ end of the RNA molecules. The y-phosphate of about 3 5 % of the newly initiated molecules was removed during the incubation period : thus upon RNAase digestion 65% of the 32P radioactivity will appear in a nucleoside tetraphosphate as pppAp or pppGp and 35% will appear in a nucleoside triphosphate as ppAp or ppGp. Treatment with PI nuclease

would yield the nucleoside tri and diphosphates, respectively. Two additional tests substantiated this interpretation. When the RNAase digest (Fig. 5 B) was treated with bacterial alkaline phosphatase, all 32P radioactivity was released as free phosphate showing that the peaks observed in Fig.3A did not contain ‘cap’ structures. When the RNAase digest was treated with nucleotide pyrophosphatase all 32P radioactivity was released as free phosphate (40 %) and pyrophosphate (60 %). No radioactivity was found in the region of nucleoside monophosphates, excluding the possibility that 32Pwas in the a-position.

Size of RNA Synthesized in vitro in Isolated Nuclei The size of labeled RNA synthesized in vitro was measured using acrylamide/agarose gels, 80 - 90 % of the 3H-labeled RNA was larger than 10 S. However,

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most of the 32P-labeled material (labeled either with [P-32P]GTP or [/3-32P]ATPmigrated in the range of 4 - 5 S (Fig. 6 A and D). The sensitivity to low (2 pg/ml) and high (200 pg/ml) concentration of a-amanitin is also shown. While most (70 - 80 %) of the 3H-labeled RNA was inhibited by 2 pg/ml a-amanitin, only very little if any of the incorporation of P-labeled triphosphates was sensitive to this concentration of the drug (Fig.6B, E). Most of the 32Pincorporation was inhibited by 200 pg/ml a-amanitin (Fig.6C, F) indicating again that they are synthesized by RNA polymerase 111. A residual fraction of both 3H and 3 2 was ~ resistant even to 200 pg/ml of amanitin, indicating that they were possibly initiated by RNA polymerase I. DISCUSSION Analysis of the RNA molecules synthesized in vitro using [P-32P]ATPor [p-32P]GTPshows that they are indeed incorporated exclusively into the 5’ ends of the newly synthesized chains. Although most of the y-position is intact under these conditions, a significant fraction (35%) of the y-phosphate still appears to be lost during the incubation period (Fig. 5). We therefore believe that the use of the p-labeled triphosphates is superior when quantitative determination of chain initiation is required. The convenient procedure for the quantification of RNA chain initiation described here, was used to discover conditions where maximal initiation could be observed. It appears that the optimal conditions for elongation are quite different from those which are optimal for chain initiation. Under the standard conditions of incubation, initiation using [fl-32P]GTP was found to be maximal over a range of 0.1 - 0.15 M ionic strength, and that of [P-32P]ATPwas much less affected by salt, while elongation still proceeded even at a much higher salt concentration (Fig. 3). Most of the RNA chains initiated in vitro are the product of RNA polymerase 111, and only very few if any were initiated by RNA polymerase 11. However, in some of our earlier experiments 20 - 50 % of the incorporation of P-32P-labeled GTP was consistently inhibited by a low concentration of aamanitin (E. Gilboa and H. Aviv, unpublished results). But in those experiments the level of activity of the nuclei was about 1/10 the activity measured in the experiments described in Fig. 3 and Table 1. We feel that these differences are real, and probably represent different types off nuclei preparations and perhaps also represent cells in different physiological conditions. But we can not yet clearly define these differences in a reproducible and meaningful manner. It is not clear whether the low level of initiation observed in vitro with regard to RNA polymerase I1 is due to some deficiency in the nuclear RNA-synthe-

sizing system or is below the limit of our detection method, which is a small percentage. We do not know, for example, the expected fraction of active genes coding for mRNA-like molecules. Indeed, Scbmincke et al. have observed that in vivo only as little as 2 - 3 % of the molecules having a 5’-triphosphate end after labeling whole cells with [32P]orthophosphate for 40min, were in hnRNA [20]. It is obvious that the detection method used (incorporation of p-labeled GTP and ATP into RNA) is biased in favour of detecting the smaller molecules. Thus, if the rate of elongation in vitro is about 3-10 nucleotides/s [lo] then 2 - 6 molecules of chain length of 100 nucleotides will be synthesized every minute, but much less than this number of molecules would be synthesized every minute if the molecules are longer. These considerations should be taken into account when developing new approaches for assaying polymerase I1 activity. The fraction of 4 - 5 3 RNA chains initiated in vitro can be calculated. From the measurements described (Fig. 6 B and D) it appears that at least SO % and probably as much as 80% of the 4--5-S RNA chains synthesized were initiated in vitro. Similar results were obtained by Udvardy and Seifart [lo]. We could also calculate the rate by which the small RNA molecules are synthesized in these nuclei. The number of molecules initiated with guanosine and adenosine triphosphates, which are synthesized every minute per nucleus, is about 100. If the rate of chain propagation in vitro is about 3 - 10 nucleotides/s [lo], then 2-6 molecules/min of chain length of 100 nucleotides can be synthesized. Assuming that all of the nuclei are equally active in chain initiation (data are not available on this point) we are led to believe that while several thousands of genes for 4- 5-S RNA are present in the cells (see note in [9]), only a small fraction of them are actively transcribed in vitro. We believe that the approach described here will be of value in identifying the factors involved in regulation of transcription. It is our pleasure to thank Drs U. Z. Littauer, M. Revel, Y . Groner & M. Edelman for helpful suggestions. The work was supported by a Contract NO1 CP 33220 from the U.S.National Cancer Institute and by the USjIsrael Binational Science Foundation.

REFERENCES 1. Chambon, P., Gissinger, F., Kedinger, C., Mandel, J. L. & Meilhac, M. (1973) in The Cell Nucleus (Bush, H., ed.) vol. 111, p. 269, Academic Press, New York. 2. Chamberlin. M. J. (1974) Annu. Rev. Biochem. 43, 721. 3. Britten, R. J. & Davidson, E. H. (1969) Science (Wash., D.C.) 165, 349- 357. 4. Davidson, E. H. & Britten, R. J. (1973) Quart. Rev. Biol. 48 565 - 61 3. 5. Reeder, R. H. (1973) J . Mol. Biol. 80, 229-241. 6. Wilson, G. N., Steggles, A. W. & Nienhuis, A. W. (1975) Pror. Natl Acad. Sci. U.S.A. 72, 4835 4839. -

400 7. Reeder, R. H . & Roeder, R. G. (1972) J . Mol. Biol. 67, 433441. 8. Gilboa, E. & Aviv, H. (1976) Cell, 7, 567-573. 9. Marzluff, W. F., Murphy, E. C. & Huang, R. C. C. (1974) Biochemistry, 13, 3689- 3696. 10. Udvary, A. & Seifart, K. H. (1976) Eur. J . Biochem. 62, 353363. 11. Furuichi, Y., Mutukrishnan, S., Tomdsz, J. & Shatkin, A. J. (1976) J . Bid. Chem. 251, 5043-5053. 12. Levin, C. J. & Zimmerman, S. B. (1976) J . B i d . Chem. 251, 1767-1774. 13. Littauer, U. Z., Kimhi, Y. & Avron, M. (1964) Anal. Biochem. 9, 85-93.

E. Gilboa, H. Soreq, and H. Aviv: Initiation of RNA Synthesis 14. Reichard, P., Elliasson, B. & Soderman, G. (1974) Proc. Natl Acad. Sci. U.S.A. 71, 4901-4905. 15. Avron, M. (1960) Biochim. Biophys. Acta, 40, 257-272. 16. Aviv, H., Voloch, Z., Bastos, R. & Levy, S. (1976) Cell, 8, 495 - 503. 17. Marzluff, W. F., Murphy, E. C. & Huang, R. C. C. (1973) Biochemistry, 12, 3440 - 3446. 18. Penman, S. (1966) J . M o l . Biol. 17, 117-130. 19. Peacock, A. C. & Dingman, C. W. (1968) Biochemistry, 7, 668 - 674. 20. Schmincke, C. D., Herrmann, K. & Hausen, P. (1976) /‘roc. NatlAcad. Sci. U . S . A . 73, 1994-1998.

E. Gilboa and H. Aviv, Department of Virology, Weizmann Institute of Science, P.O. Box 26, Rehovot, Israel

H. Soreq, Department of Neurobiology, Weizmann Institute of Science, P.O. Box 26, Rehovot, Israel