Initiation of DNA-Dependent RNA Synthesis and the Effect of Heparin on RNA Polymerase

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1 Europen J. Biochem. 3 (1967) Initition of DNA-Dependent RNA Synthesis nd the Effect of Heprin on RNA Polymerse G. WALTER, W. ZILLIG, P. PALM, nd E. FUCHS Mx-Plnck-Institut fur Biochemie, Munchen (Received July 18,1967) The kinetics of RNA synthesis, ctlyzed by DNA-dependent RNA polymerse from Escherichi coli, show lg phse t high ionic strength or t low temperture when using doublestrnded DNA s templte. This lg phse increses with decresing temperture t constnt ionic strength or with incresing ionic strength t constnt temperture. With single-strnded DNA s templte, no lg phse is observed. The length of the lg phse is gretly shortened by preincubtion of the enzyme with DNA t low Mg++ concentrtion, independent of the presence of substrte. Heprin is strong inhibitor of free nd DNA-bound polymerse. Two heprin molecules re required for the inctivtion of one enzyme molecule. Enzyme engged in RNA synthesis is instntly inhibited on single-strnded DNA but not on double-strnded DNA. With the id of this inhibitor it is shown tht 10minutes fter the strt of incubtion, t 16, ll enzyme molecules hve initited RNA chins on double-strnded DNA. The synthesis of RNA by DNA-dependent RNA polymerse is complex process, in which severl consecutive phses my be distinguished. The first step, the binding of the free enzyme molecules to specific sites on the DNA, is fst, reversible event [l-51. The second step is the initition of RNA synthesis, followed by the synthetic rection itself. The finl step is the relese of product from the templte. The second nd third step consist of severl events, which theoreticlly must be postulted, but experimentlly re not sufficiently differentited. At the site of trnscription there should occur loclized melting of the DNA double helix to permit bse piring of the ribonucleoside triphosphtes with the codogenic strnd. While the enzyme progressively trnscribes the templte there is constnt number of nucleotides t the growing end of the RNA chin, in the form of hybrid with the codogenic strnd of the DNA. On the end of this hybrid region the RNA strnd must be replced by the non-codogenic DNA strnd [6]. On double-strnded DNA templte the synthesis phse begins with lg [7] which is not detectble t 37 under our stndrd conditions but is very pronounced t lower tempertures or t high ionic strength. The results presented in this pper show tht this lg phse is the expression of rtelimiting event which occurs between the binding of Enzymes. DNA-dependent RNA-polymerse or nucleosidetriphosphte: RNA nucleobidyltrnsferse (EC ) ; pyruvte kinse (EC ). the enzyme to the DNA nd the ctul synthetic phse. The results presented in this pper hve been reported t the Fruhjhrstgung der Gesellschft fur Biologische Chemie, Tubingen, April, 4th nd 5th) MATERIALS AND METHODS Preprtion of RNA Polymerse DNA-dependent RNA polymerse ws prepred from E. coli K12 Hfr s previously described [8,9]. RNA Synthesis Assy RNA synthesis ws mesured by the incorportion of [8-14C]ATP (0.1-1.O C/mole) into trichlorocetic cid-insoluble mteril s described before [8,9]. The incubtion conditions (temperture, substrte- nd slt concentrtions nd the kind of the templte) re described in the legends. Biochemicls [8-14C]ATP ws purchsed from Schwrz-Bio- Reserch, Inc., unlbelled ribonucleoside triphosphtes from P-L Biochemicls, Inc., sucrose ( enzyme grde ) from Mnn Reserch Lbortories, Inc., nd heprin (100 I.U./mg) from Chemische Fbrik Promont GmbH, Hmburg. DNA from phges T4, M13, QX174 nd il ws prepred s described in the previous pper [9]. Clf Thymus DNA ws obtined from Sigm Chemicl Compny.

2 Vol. 3, No. 2,1967 G. WALTER, W. ZILLIG, P. PALM, nd E. FUOHS 195 RESULTS DETERMINATION OF THE LENGTH OF THE LAG PHASE The lg phse is followed t low temperture by phse of nerly constnt rte of RNA synthesis. The point where the increse of product becomes liner is difficult to determine with sufficient ccurcy. Therefore, s convenient mesure for the length of the lg phse, tlp, the time is used where the extrpolted liner portion of the curve intercepts the time xis. 1 1 log ~ ginst -, where T is the bsolute tempertlp T ture (Fig.2). This plot shows two liner portions 30 A - E In N? E 2c - n W I- [L 0 [L 0 z 10 3 j /r Fig.2. Arrhenius plot of log(l/tlf) versus l/t. The vlues for log (l/tlp) hve been clculted from the experiment described in Fig. 1, i TIME OF INCUBATION (mid Fig. 1. Kinetics o,f RNA synthesis t vrious tempertures. The rection components were t the following concentrtions : T4 DNA, 0.1 mg/ml; CTP, GTP, UTP nd [8-14C]ATP ech t 1 mm; Mg++cette, 30 mm; NH,CI, 0.13 M; Tris cette ph 7.9,0.03 M; RNA polymerse 68 pg (78 units)/ml. Sufficient mounts of the incubtion mixtures were incubted t ech temperture. For the ssy of RJNA synthesis liquots were removed t the times indicted s described in Methods INFLUENCE OF DIFFERENT PARAMETERS ON THE LENGTH OF THE LAG PHASE Dependence of the Length of Ihg Phse on Temperture Under our stndrd conditions (0.03 M Mg++, 0.13M NH,CI), the lg phse is only detectble when the rection kinetics re followed t sufficiently low temperture (Fig. 1). It is not detectble t 37" but then grdully ppers with flling temperture, incresing rpidly below 17". The lg phse is very likely the result of rtc determining rection preceding the phse of RNA synthesis. The ctivtion energy, AH, for this rection my be clculted from n Arrhenius plot of 131 I I I I in Fig.3. Arrhenius plot of the logrithm of the mximl rte of RNA synthesis versus the reciprocl of the temperture. The vlues for the logrithms of the mximl rte of RNA synthesis hve been clculted from Fig. 1 with chnge of slope occuring t vlue corresponding to bout 17". From the slope below this temperture the vlue for AH is bout 180 kcl; from the slope bove this temperture it is bout 30 kcl/mole. The slopes of the liner portions of the time curves of RNA synthesis (Fig. 1) represent the mximl rtes of RNA synthesis themselves, independent of the event cusing the lg phse. From the Arrhenius plot of these dt (Fig.3) n ctivtion energy

3 196 Initition of RNA Synthesis Europen J. Biochem. (AH) of 23 kcl/mole hs been clculted for the synthesis rection. This vlue is considerbly lower thn tht obtined for the "lg phse event", s is expected when the rection cusing the lg phse is rte determining for the initition of RNA synthesis t low temperture. Dependence of the Length of the Lg Phse on the Concentrtions of Enzyme nd T4-DNA In Fig.4 the kinetics of RNA synthesis for 3 different rection mixtures re shown, DNA lwys in exccss. In the one cse the enzyme concentrtion is hlf tht of the control, in the other both enzymend DNA concentrtion re hlf tht of the control mixture. In ll cses liquots hve been tken which re both lowered by fctor of 5, so tht the collision frequency is lowered by fctor of 25, the lg phse is only slightly incresed (1.4-fold). From these results it follows tht the initil binding of enzyme to DNA is very fst rection which cnnot be the cuse of the lg phse. Effect of Pre-Incubtion of Vrious Rection Components on the Lg Phse When enzyme nd substrte re preincubted for 10minutes t 37" in complete rection mixture minus DNA nd the rection is strted t 15" by I I PREINCUBATION WITH: TIME OF INCUBATION (rnin) F'ig.4. Kinetics of RNA synthesis ut 15' t different DNA nd polymerse concentrtions. Three different incubtion mixtures contined: (A) 112 yg (100 units) poiymerse/ml nd mg T4 DNA/ml; (0) 56 pg (50 units) polymerse/ml nd mg T4 DNA/ml nd (0) 56 pg (50 units) polymerse/ml nd mg T4 DNA/ml; ll other components of the incubtion mixture were t the sme concentrtions s in Fig.1. In ll three cses liquots contining the sme mount of enzyme were removed t the times indicted: (A) 0.25 ml, (0) 0.5 ml, (0) 0.5 ml. The ordinte gives the incorportion of nmoles AMP in these liquots TIME OF INCUBATION (nin) Fig.5. Effect of preincubtion of enzym,e with DNA, slt nd n incomplete mixture of substrte t 37" on the kinetics of RNA synthesis t 15". The rection mixtures contined ll the components required for RNA synthesis t the concentrtions given in Fig. 1, except tht the mixture of substrtes ws incomplete s indicted in the figure. These were preincubted for 5 minutes t 37", cooled to 15' nd strted, t t = 0 minutes, by the ddition of the lcking substrtes. Polymerse ws t 7 units/ml; the kinetics were followed.s described in Fig. 1 contin the sme mount of enzyme. As expected, fter the lg phse, when presumbly ll enzyme molecules re ctive in RNA synthesis, the mximl rection rtes re bout the sme in ll cses. Moreover, within the experimentl ccurcy, tlp stys unchnged in spite of the chnge in the number of collisions between enzyme molecules nd binding sites on the DNA which would be hlf tht of the control mixture in the one cse nd qurter tht of the control rection mixture in the other experiment. Even when enzyme- nd DNA concentrtions the ddition of DNA, the lg phse ppers in its full length. This excludes the ssumption of rte determining ctivtion of enzyme by substrte. When n incomplete mixture of ribonucleoside triphosphtes is offered in the presence of ll other rection components during pre-incubtion period of 5 minutes t 37" nd RNA synthesis is strted by the ddition of the lcking ribonucleoside triphosphtes t 15", the pprent lg phse becomes shorter in some cses nd lmost disppers in others (Pig. 5). However, certin,mount of incorportion

4 Vol.3, No.2, 1967 G. WALTER, W. ZILLIG, P. PALM, nd E. FTJCHS 197 cresed to Ol0 of its vlue without pre-incubtion (Fig.6). The sme result is obtined, when no substrte is present t low Mg++concentrtion (0.2 mm) during the pre-incubtion period. Therefore, the decrese of tlp must be function of the Mg++concentrtion. / Effect of substrte Concentrtion on the Length of the Lg Phse A vrition in the substrte concentrtion from substrte sturtion, 1 mm for ech of the 4 ribonucleosidetriphosphtes, to 1/50 of this vlue does not cuse significnt ltertion of tlp. I, I I I TIME OF INCUBATION (mid Fig.6. Effect of pre-incubtion of polymerse with DNA, nd vrious other components t 37", on the initition of RNA synthesis t 15". During pre-incubtion period of 10 minutes t 37", the rection mixtures contined ll the components of norml mixture (Fig. 1) but lcking (0) enzyme, (0) substrtes, ( x ) Mg++ cette, the smll mount of Mg++ cette due to the ddition of enzyme, 0.2 mm, ws neutrlized by 0.2 mm EDTA, (v) Mg++cette, nd (A) Mg++cette nd substrtes. In the lst two mixtures 0.2 mm Mg++ cette ws present during pre-incubtion due to the ddition of enzyme. After cooling to 15" the mixtures were strted by the ddition of the lcking components. Enzyme ws present in concentrtion of 150 pg (16 units)/ml; the kinetics were followed s described in Fig. 1 When enzyme nd DNA re pre-incubted for 10 minutes t 37" t stndrd slt concentrtion (0.03 M Mg cette, 0.13 M NH4C1) nd the rection is strted t 15" by the ddition of substrte, the lg phse ppers in full length. This gin excludes the ide tht the primry binding rection, which occurs under these conditions, is the cuse of the lg phse. When, however, enzyme, DNA, substrtes nd NH4C1 (0.13 M) re pre-incubted for 10 minutes t 37" in the bsence of Mg++ ions or in the presence of n mount of Mg++ insufficient to llow synthesis (0.2 mm) nd the rections is strted, t 15", by the ddition of sufficient Mg++ (0.03 M), tlp is de- u [MgACETATEl(M) Fig. 7. Effect of Mg++ cette concentrtion on tlf. Conditions were the sme s in Fig. 1 except tht the incubtion temperture ws 37' nd the Mg++ cette concentrtion ws vried s indicted in the figure; polymerse ws t concentrtion of 115 pg (74 units)/ml Effect of Slt Concentrtion on the Length of the Lg Phse tlp increses with incresing concentrtion of Mg++ s well s of NH4+ ions. In Fig.7 the vlues of tlp t 37" re plotted ginst the corresponding Mg++ concentrtions. If tlp is used s n inverse mesure of the velocity of the lg phse rection, it is seen tht the rte of this rection, bove concentrtion of 0.04 M Mg++, decreses steeply. When the NH4C1 concentrtion is vried t constnt Mg++concentrtion the result is qulittively similr. Dependence of the Lg Phse on the Nture of the Templte The lg phse is lwys observed when doublestrnded DNA is used s templte (DNA of phge T4, phge A, clf thymus, EhrLich scites tumor cells, Scchromyces cerevisie). It is not detected t ll

5 198 Initition of RNA Synthesis Europen J. Biochem. with single-strnded DNAs such s dentured T4- DNA, nd DNAs of nd MI3 (Fig.8). It seems, therefore, tht the rection which is rte limiting for the initition ofrna synthesis on doublestrnded templte DNA is not required when singlestrnded DNA is the templte. ANALYSIS OF THE INITIATION OF RNA SYNTHESIS BY THE USE OF HEPARIN Inhibition of RNA Synthesis by Heprin Like other polynions, heprin is n inhibitor of RNA synthesis by DNA-dependent RNA polymerse. The inhibition cused by heprin rl0,iil is much stronger thn tht cused by RNA [12,13] of RNA synthesis on double-strnded DNA t 37", no inhibition is observed during the period immeditely following the ddition [9]. Therefore, enzyme engged in RNA synthesis on double-strnded DNA is not blocked by heprin, in contrst to free enzyme or DNA-bound enzyme which hs not strted synthesizing RNA. On the other hnd, enzyme which is engged in RNA synthesis on single-strnded DNA is inhibited lmost instntneously t ny time during the course of the rection (Fig. 9). Amount of Heprin Required for Inhibition of RNA Synthesis The mount of heprin which is required for the inhibition is not relted to the quntity of DNA 1 I present in the incubtion mixture but depends on the mount of enzyme which is used. The quntittive I I I HEPARIN 25 AT rnin TIME OF INCUBATION (mid Fig.8. Influence of the DNA templte on the kinetics of RNA synthesis. Conditions were t,he sme s in Fig.1 with the following vritions: s templtes were used (0) ntive T4 DNA, 0.05 mg/ml; (0) dentured T4 DNA (5 minutes, loo"), 0.05 mg/ml; (A) 0174 DNA, 0.05 mg/ml nd (v) Mi3 DNA, O.lmg/ml. With the first three templtes the incubtion temperture ws 16' nd the enzyme concentrtion ws 218 pg (254 units)/ml; with the lst one the t.emperture ws 15" nd the enzyme ws t 102 pg (90 units)/ml Totl inhibition, even t very low concentrtions of the inhibitor, is observed when the polynion is dded to the free enzyme before the other components, nd lso when the enzyme is first preincubted with DNA nd then the substrtes re dded together with the polynion. Therefore, s heprin inctivtes not only free enzyme, but lso enzyme ttched to the DNA in the primry binding rection, it hs higher ffinity thn DNA for the polymerse. When the inhibitor is dded severl minutes fter the initition TIME OF INCUBATION (min) Fig.9. Heprin inhibition of RNA synthesis on templte DNA from phge QX174. The conditions were the sme s in Fig. 1 except tht NH,CI ws 0.1 M DNA, 0.1 mg/ml, ws used s templte; the polymerse concentrtion ws 57 pg (40 units)/ml; the incubtion temperture ws 37"; heprin ws dded t finl concentrtion of 20pg/ml t, the times indicted on the figure correltion between the residul RNA synthesis nd the mount of heprin dded is shown in Fig. 10. In this experiment the enzyme ws first pre-incubted with the inhibitor for I0 minutes t 37". Then the rection ws strted by the ddition of DNA nd the incubtion continued for lominutes t 37". The kinetics of the inctivtion rection re rpid. With n mount of heprin sufficient for hlf inhibition the inctivtion is complete fter 5 minutes t 37". The molr rtio of heprin/enzyme required for 90 inhibition of RNA synthesis is 2, clculted using moleculr weight of 12,500 for the heprin prepr-

6 Vl. 3, No. 2, 1967 G. WALTER, W. ZILLIG, P. PALM, nd E. FUCHS 199 tion [14,15] nd moleculr weight of 720,000 for the enzyme prticle [lo] nd ssuming tht ll enzyme molecules re ctive. At very low heprin concentrtions n ctivtion of RNA synthesis is observed. A significnt inhibition of ctivity begins t molr rtio of heprin to enzyme of bout The theoreticl curve in Fig. 10 hs been clculted ssuming tht 2 heprin molecules inctivte 1 polymerse molecule in n irreversible rection, nd tht one heprin molecule per polymerse prticle hs no effect on the ctivity. [HEPARIN] (pg/02ml) Fig.10. Dependence of the rte of RNA synthesis on the concentrtion of heprin. The enzyme, 102 pg (90 units)/ml, ws pre-incubted for 10 minutes t 37, with vrying mounts of heprin in the presence of ll other rection components, except the DNA, in the concentrtions given in Fig. 1. The rection ws strted t the sme temperture by the ddition of DNA. Incubtion time ws 10 minutes for ech smple. The upper curve is n verge of 4 experiments. The lower theoreticl curve ws clculted from the following ssumptions: () two heprin molecules (Mol. wt. 12,500) irreversibly combine with one polymerse prticle (Mol. wt. 720,000) to form n inctive complex; (b) one heprin molecule per polymerse prticle binds to the enzyme but neither inctivtes nor ctivtes it; (c) the enzyme consists of ctive prticles only. The number of free enzyme molecules (Z,,), molecules with one (2,) nd with two bound heprin molecules (2,). -. hve been clculted for ech rtio of heprin to polymerse from the equtions: 2, = (22-ivy N2 42 nd Z,= -, where 2 is the toti N(2.Z- N) 2, = number of polymerse molecules nd N the totl number of heprin molecules Physicl Interction of RNA-Polymerse Prticles with Heprin When preprtion of RNA-polymerse consisting minly of 24s-prticles [16] is treted with n excess of heprin (more thn two molecules of heprin per polymerse molecule) quntittive trnsformtion into mteril sedimenting round 15s is observed. When one heprin molecule per enzyme prticle is dded, bout hlf of the 24sprticles dispper coincident with the formtion of this new moleculr form. The moleculr weight of this form hs not yet been determined. However, electron microscopy suggest tht the rection does not led to disggregtion of the enzyme prticle. Use of Heprin for Anlysis of the Lg Phse in RNA Xynthesis Since heprin inhibits lmost instntneously free or DNA-bound polymerse but not enzyme engged in RNA synthesis, it is suitble gent for the investigtion of the kinetics of initition of RNA synthesis. Fig. 11 describes the influence of heprin on the kinetics t 16. The norml time course of the rection (closed circles) hs lg phse (tlp) of bout 3 minutes. The curve connecting the open circles ( heprin curve ) shows the susceptibility of RNA synthesis to heprin inhibition t vrious periods during the time course. It ws obtined by first strting synthesis s in the control, dding n excess of heprin t vrious times therefter nd incubting further for 15 minutes. From this curve it cn be seen tht heprin inhibits RNA synthesis most strongly during the erly periods due to its inhibition of the free or DNA-bound enzyme. Its ction is seen more clerly from the slopes of the stright lines interconnecting the norml nd heprin curve for the respective 15 minutes incubtion periods with heprin. The slopes of these lines re rough mesure of the frction of enzymtic ctivity tht is insensitive to inhibition by heprin during this intervl. Becuse of the dependence of the slope of these lines on the shpe of the norml curve, more correct mesure for the frction of enzymtic ctivity insensitive to heprin inhibition is the rtio of the mount of RNA synthesized during the 15-minute intervls in the presence of heprin to tht in its bsence. This rtio is plotted s function of time in Fig.ll (crosses). Here it is clerly seen tht the frction of enzymic ctivity which is insensitive to heprin rises rpidly during the first 10 minutes of incubtion. It reches plteu vlue of bout 0.83 during the liner phse of RNA synthesis nd then finlly declines slowly s the rte of synthesis decreses. DISCUSSION The synthesis of RNA by DNA-dependent RNA polymerse strts with pronounced lg phse t low temperture or t high ionic strength. The lg phse is the expression of rte limiting step in the initition of the synthetic rection. The only rection which is known t this time to proceed the beginning

7 200 Initition of RNA Synthesis Europeii J. Biochem E , v n W G LT 10 x LT 0 V z - 5 I Q I I I I I I I I TIME OF INCUBATION (mid Fig. 11. Influence of heprin, dded t vrious times fter the strt of incubtion, on the time course of RNA synthesis. Conditions re the sme 8s in Fig. 1, except tht the polymerse concentrtion ws 218 pg (254 units)/ml, nd the T4 DNA concentrtion 0.05 mg/ml. Incubtion temperture ws 16. From lrge incubtion mixture liquots were removed t vrious times fter the strt of the rection nd either () directly prepred for counting (norml kinetic) (0) or (b) pipetted into 2.5 vg heprin in 0.01 ml wter, then further incubted for 15 minutes nd prepred for counting. Control liquots were pipetted into the sme mount of wter without heprin nd further incubted for 15 minutes. The heprin curve (0) hs been obtined by dding the difference between the norml kinetics nd the wter controls to the incorportion vlues obtined with heprin. The time difference between points on the two curves connected by stright lines is 15 minutes. The rtios of the incorportion in 15 minutes in the presence of heprin (tken from these lines) to the incorportion in 15 minutes in the bsence of heprin (tken from the norml curve for the sme time intervl) re plotted in the upper curve ( x ) of synthesis is the reversible binding of the enzyme to the templte DNA. If the lg phse would be cused by this binding rection, its length should depend on the concentrtions of enzyme nd DNA in the rection mixture. Since this is not observed, nother rection must be postulted for the lg phse. Since tlp remins unchnged when the synthesis is initited by the ddition of DNA to pre-incubted mixture of the enzyme nd ll other rection components, rection between enzyme nd substrte or slt cnnot be the cuse of the lg phse. One therefore hs to serch for n event which occurs between the binding of the enzyme to the DNA nd the beginning of the synthetic rection. Severl independent observtions support the theory tht the lg phse is the consequence of limited strnd seprtion rection which is cused by the enzyme on the DNA t the site where RNA synthesis is initited : ) At constnt ionic strength tlp increses with decresing temperture, rising steeply below 17. This reltionship bers close similrity to prt of DNA melting curve nd could result from the temperture dependent melting of smll prt of the DNA double-strnd under the influence of the enzyme molecule. b) At constnt temperture the length of the lg phse increses steeply bove certin ionic strength suggesting tht bove this slt concentrtion the melting of smll prt of the DNA double-strnd by the enzyme molecule requires high energy of ctivtion. c) When single-strnded DNA is used s templte (dentured T4-DNA nd DNA from phges OX174 nd M13) no lg phse is observed. d) The length of the lg phse is gretly shortened by pre-incubting the enzyme with DNA t low ionic strength, independent of the presence of substrte. This mens tht chemicl rection involving the substrtes cnnot be responsible for the lg phse rection nd further supports the theory tht the lg is cused by physicl process. The fct tht this reduction of the lg phse occurs only t low ionic strength suggests tht under these conditions the DNA-bound enzyme brings bout some chnge on the templte, possibly loclized melting, which is required for the initition of RNA synthesis. Such n interction would be hindered t high ionic strength. The events leding to tke initition of RNA synthesis on double-strnded templte DNA my bc formulted s follows : (I) E + DNA 2 E - DNA Binding rection (11) E - DNA?t E - DNA * Melting rection (111) E - DNA* + substrtes Mg++ -pyrophosphte + E - DNA - RNA RNA synthesis phse The first rection (I), the primry binding of the enzyme to DNA, hs been well chrcterized nd found to be reversible process whose equilibrium strongly fvors the enzyme-dna complex [l, 21. The melting rection (11) involves the formtion of the ctivted complex (E - DNA*) in which, s we ssume, the enzyme hs effected loclized seprtion of the DNA double helix t the binding site. The bility to chnge the length of the lg phse by vrying the ionic conditions during the pre-incubtion of enzyme with DNA shows tht the melting rection is reversible. The equilibrium of this rection depends on the temperture nd the ionic strength. De-

8 V01.3, Y0.2, 1967 G. WALTER, W. ZILLTG, P. PALM, nd E. FUCHS 201 cresing thc temperture nd incresing the ionic strength shifts it to the left, thereby incresing tlp. The lst rection (IIl), the RNA synthesis phse, removes the ctivted complex from the equilibrium of the melting rection with the concomitnt formtion of RNA product,. This scheme corresponds formlly to the sequence of rections postulted by Anthony, Zeszotek nd Goldthwit [17] for the initition of RNA synthesis. The second step in the rection sequence discussed by these uthors nd termed initition hs been interpreted s the formtion of DNA-enzymepurine nucleotide complex. In our scheme, the corresponding step, termed the melting rection, involves the formtion of n ctivted enzyme-dna coniplex precceding the beginning of RNA synthesis. The inittion complex, s isolted by Anthony rid coworkers fter pre-incubtion in the prescncc of n incomplete mixture of substrtes, could be complex of DNA, enzyme nd short nucleotide sequences. This interprettion is supported by thc finding of polynucleotide synthesis in the presence of n incomplete mixture of substrtes (see Fig.5). Our results show tht the length of the lg phse is independent of the concentrtion of substrtes over wide rnge nd tht the rection cusing the lg phse cn be seprted from the phse of RNA synthesis. Heprin hs proved to be useful gent in mlyzing the initil phse of RNA synthesis. Since it inhibits free or DNA-bound but not synthesizing enzyme, it enbles the determintion of the frction of polymerse which hs initited RNA synthesis t ny moment during the rection. These experiments (Fig.11) show tht the frction of polymerse noninhibited by heprin reches plteu t 10 minutes. This mens tht t this time ll enzyme molecules hve initited RNA synthesis. Similr results hve been obtined by mesuring the kinetics of the ppernce of y-32p-lbeled 5 -triphosphte end groups in the synthesized RNA [IS]. Since synthesizing enzyme is not inhibited by heprin one would expect, therefore, tht the heprin curve nd the control curve (Fig. 11) would become superimposble fter 25 minutes. Moreover the frction of enzymtic ctivity resistnt to heprin would be expected to rech vlue of 1 fter 10 minutes. Tht this is not the cse is very likely due to reversiblc first order inctivtion of the synthesizing enzyme itself, which is mde irreversible by heprin [9]. Heprin hs been shown to interct physiclly with free RNA-polymerse nd to inctivte it t rtio of two inhibitor molecules per enzyme molecule; this indictes tht there re two sites of binding for the polynion on the 24s-prticle. The theoreticl curve in Fig. 10 hs been clculted under the ssumption tht 1 inhibitor molecule per polymerse prticle does not influence the enzyme ctivity nd two inhibitor molecules per enzyme molecule led to totl inctivtion. Tht the experimentl curve (Fig. 10) linost prllels the theoreticl one, supports this conclusion. The prllel shift of the experimentl curve long the bsciss is cused by the ctivtion of RNA synthesis observed t very low heprin concentrtions. This might be explined by ssuming tht smll mount of heprin is required to neutrlize n inhibitor in the enzyme preprtion or tht the heprin preprtion contins n enzyme ctivtor. Since enzyme synthesizing RNA on doublestrnded DNA is not inhibited by heprin, both sites on the enzyme molecule which potentilly bind heprin re involved in RNA synthesis nd re thus protected ginst the ction of the inhibitor. On thc other hnd RNA synthesis on single-strnded DNA is blocked lmost spontneously by heprin. This is plusible if one ssumes tht, in this cse, only one of the two sites is involved in the synthetic rection nd tht the other is ccessible to the inhibitor t ny moment. Our thnks re due to Professor A. 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