Studies of the binding of Escherichia coli RNA polymerase to DNA

Studies of the binding of Escherichia coli RNA polymerase to DNA

J. Mol. Bid. (1972) 70,209-220 Studies of the Binding of Escherichia coli RNA Polymerase to DNA IV. The Effect of Rifampicin DAVID C. Hmmrf, on Bind...

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J. Mol. Bid. (1972) 70,209-220

Studies of the Binding of Escherichia coli RNA Polymerase to DNA IV. The Effect of Rifampicin DAVID C. Hmmrf,

on Binding and on RNA Chain Initiation

WALTER F. MANQEL AND MICHAEL J. CHABXBERLIN

Departments of Biochemistry and iKoleculur Biology and the Virus Laboratory University of California, Berkeley, Calij. 94720, U.S.A. (Received 21 September 1971, and in revised form 26 April 1972) Binding of Escherkhia coli RNA polymerase to T7 DNA is not prevented by complexing of the enzyme with rifampicin, nor does rifampicin increase the rate of dissociation of the highly stable RNA polymerase holoenzyme-T7 DNA complex. Both free RNA polymerase and RNA polymerase-T7 DNA complexes are attacked by rifampicin in second-order reaotions although the rate constant for the latter reaotion is reduced about loo-fold. Alteration of the stability of the holoenzyme-DNA complex does not appreciably change the rate of rifampicin attack, nor does removal of sigma subunit from the RNA polymerase-DNA complex. It is suggested that the relative resistance of RNA polymerase holoenzyme-T7 DNA complexes to attack by rifampicin, when the drug is added together with the nucleoside triphosphates, is due to the rapid rate of RNA chain initiation by this complex.

1. Introduction The antibiotic rifampicin inhibits bacterial RNA synthesis in vivo and in vitro by binding to the bacterial RNA polymerase protein (Wehrli, Knusel, Schmid t Staehelin, 1968 ; di Mauro et al., 1969). Studies of the individual steps of enzymic RNA synthesis suggest that once chain initiation has been accomplished, RNA synthesis becomes resistant to inhibition by the drug (Sippel & Hartman, 1968; So & Downey, 1970). Hence, rifampicin must act to block one of the first two steps in enzymatic RNA synthesis, template binding or chain initiation. Since binding of RNA polymerase to DNA was not blocked by rifampicin (Umezawa, Mizuno, Yamazaki & Nitta, 1968), it seemed likely that the drug acted to prevent RNA chain initiation. However, recently Sippel & Hartman (1970) have found that pre-incubation of RNA polymerase with DNA leads to the formation of a binary complex, which is able to initiate RNA synthesis in the presence of rifampicin. We have proposed that initiation of specific T7 RNA chains by RNA polymerase holoenzyme involves the prior formation of a highly stable complex between RNA polymerase holoenzyme and T7 DNA, which occurs during the template binding stage of the reaction (Hinkle & Chamberlin, 1970). By this model, the sequence of steps in site selection by RNA polymerase involves (1) reversible, weak binding to t Present address: Department 02116, U.S.A.

of Biological

Chemistry, 209

Harvard

Mediaal School, Boston,

Mess.

210

D. C. HINKLE,

W. F. MANGEL

AND M. J. CHAMBERLIN

random sites on DNA, leading ultimately to (2) attachment to a site at or near the specific promoter site on the T7 molecule and (3) a reaction to form a highly stable complex. Because of the high temperature coefficient of reaction (3), it has been suggested that the reaction leads to separation of the DNA strands at or near the promoter site (Hinkle & Chamberlin, 1970; Zillig et al., 1970). Thus, rifampicin might act to block one of these specific steps in the formation of the specific complex between

holoenzyme and the T7 promoter site. In this report, we show that rifampicin does not block the formation of the highly stable complex between RNA polymerase holoenzyme and T7 DNA, nor does the attachment of rifampicin to the enzyme increase the rate of dissociation of this complex.

RNA

polymerase

holoenzyme

bound in this complex

is attacked

by rifam-

picin in a second-order reaction with a rate constant about 100 times lower than that of free RNA polymerase. Complexes between core polymerase and T7 DNA are attacked at an identical rate. The ability of RNA polymerase holoenzyme bound to T7 DNA to initiate RNA chains in the presence of the drug depends on the rapid conversion of the enzyme to a drug-resistant form through RNA chain initiation.

2. Materials and Methods RNA polymerase holoenzyme (fraction 6) and core polymer&se (fraction 7B) were purified from E. coli B or K12 using a modification (Mange1 & Chamberlin, manuscript in preparation) of the method of Berg, Barrett t Chamberlin (1971). Procedures used to assay for enzymic activity (Berg el al., 1971) and for binding of RNA polymerase to 3H-labeled T7 DNA using the nitrocellulose filter assay (Hinkle & Cbamberlin, 1972a) have been described elsewhere. Assay solution B contains 0.2 Irr-Tris (pH 8), 0.1 M-MgCl,, 0.06 M-2-meroaptoethanol and 5 mg of bovine serum albumin/ml. RNA polymeraae is diluted in a solution containing 0.05 ma-Tris (pH 8), 6 b-2-mercaptoetbanol, 0.6 mu-EDTA and 2 mg BSA/ml.t. Binding buffer containz 0.01 M-Tris.HCl (pH 8), O*Ol M-MgCl,, 0.01 Y2-mercaptoethanol, 0.001 X-EDTA and 0.06 rd-NaCl. Rifampicin (Rifampin) was donated by the CIBA Pharmaceutical Co. and was prepared as an aqueous solution. A molecular weight of 823 daltons w&s assumed for rifampicin.

3. Results (a) Effect of tifa?rz@cin on the binding of RNA polymerme to T7 DNA It has been reported by Umezawa et al. (1968) that rifampicin does not prevent the binding of RNA polymerase to T7 DNA. Using the nitrocellulose titer technique to assay complex formation (Hinkle & Chamber& 1972a) we also find that the drug has no effect on the formation of a non-filterable complex (Table 1). However, it seemed possible that rifampicin might block formation of the highly stable complex (Hinkle & Chamberlin, 1970) between RNA polymerase holoenzyme and T7 DNA, either through failure of the rifampiciu-enzyme complex to bind to the promoter site, or as a result of mobilization of this complex after it had formed. Preliminary experiments (Table l), in which the complex formed between 3H-labeled T7 DNA and the holoenzyme in the presence of rifampicin was diluted with cold DNA and incubated for ten minutes, showed that rifampicin did not lead to rapid dissociation of the RNA polymerase holoenzyme from DNA. This result, taken with the rapid rate of attack of rifampicin on the holoenzyme-T7 DNA complex (Sippel & Hartman, 1970, and below), indicated that the RNA polymerase is attacked by the drug while t Abbreviation

used: BSA, bovine serum albumin.

INTERACTION

OF RIFAMPICIN

AND

RNA

211

POLYMERASE

TAEZE 1 Effect of rifampicin

on formation

Conditions

No rifampiain 2 118rifampioin/ml.

of the highly stable RNA polym.erase-T7 DNA mph Retention

Before unlabeled

of 3H-labeled DNA

T7 DNA

After

(%)

unlabeled

83 64

DNA

70 64

Reactions (0.10 ml.) contained binding buffer, 60 H of BSA, 1 nmole 3H-lebeled T7 DNA (10,000 cts/min), 0.06 m of RNA polymerase holoenzyme (fraotion 6,16,000 d(A-T) unit&q) and O-2 H rifampioin where indiaated. After 10 min at 37°C the amount of 3H-labeled T7 DNA complexed with RNA polymerase was determined by filtration. To determine the rate of dissooiation of the enzyme-DNA oomplex, 34 nmoles of u&beled T7 DNA were added to the reaotion and incubation continued for 10 min at 37°C before filtration.

bound to DNA and that dissociation to give free enzyme is not involved in the inactivation of the binary complex as had been suggested (Bautz & Bautz, 197Ou,b). The rate of dissociation of the RNA polymerase holoenzymeT7 DNA complex is so slow under these conditions (tt about 60 hr) that it would be difficult to detect even a twofold increase in the rate of dissociation (Hinkle & Chamberlin, 1972a). However, it is important to know if rifampicin affects the stability of the RNA polymerase holoenzyme complex in a quantitatively significant way, as it must do if it acts to block a step in site selection. To determine the effect of rifampicin on dissociation of the holoenzyme-DNA complex most sensitively, the rate of dissociation was measured in 0.1 M-NaCl. At this salt concentration, dissociation is more rapid and a more valid comparison of the rates of dissociation of the holoenzyme and holoenzyme-rifampicin complex from DNA can be obtained (Fig. 1). The results reveal no detectable difference in the rates of dissociation of complexes formed in the presence or in the absence of rifampicin. We conclude that rifampicin does not detectably affect the formation or the dissociation of the highly stable complex between E. wli RNA polymerase and T7 DNA. (b) Kinetics

of inactivation

of RNA polymerase h&enzyme

by tijampicin

The fact that the binding of rifampicin to RNA polymerase does not prevent the formation of a highly stable enzyme-DNA complex suggested that the formation of this complex would not prevent the binding of rifampicin to RNA polymerase. We wished to compare the rate of inactivation by rifampicin of free RNA polymerase to that of RNA polymerase bound to T7 DNA. To determine the parameters for the reaction of rifampicin with free RNA polymerase, samples of RNA polymerase holoenzyme were incubated in the presence or absence of rifampicin. At intervals, portions were withdrawn and added to a mixture of T7 DNA and ribonucleoside triphosphates. Because of the rapid rate of binding of RNA polymerase to DNA (Hinkle & Chamberlin, 19728) and the large increase in resistance to thermal inactivation and to rifampicin attaok conferred by binding to DNA, this sampling can be considered to rapidly convert the enzyme to a stable form for assay. The kinetios of inactivation of free RNA polymerase obtained under these conditions are shown in Figure 2. Under standard assay conditions but in the absence of DNA or nucleoside

212

D. C. HINKLE,

W. F. MANGEL

F n

AND

M. J. CHAMBERLlN

60 t

j,j

I ,

0

I Time

,

j

2

3

(hrl

FIG. 1. Effect of rifampioin on the kinetics of diasooiation of the highly stable RNA polymeram holoenzyme-T7 DNA complex. Reactions contained 0.27 erg of RNA polymeraae holoeneyme (fraction 6, 24,000 d(A-T) units/mg), 6 nmoles of 3H-labeled T7 DNA, 260 erg BSA, 26 qnoles NaCl (a final NaCl concentration of 0.1 M) and 0.36 ml. of binding buffer in a final volume of 94 ml. Where indicated, 2 pg of rifampioin were also added. After incubation for 10 min at 37”C, 260 nmoles of unlabeled T7 DNA was added in 0.1 ml. of binding buffer (zero time) and inoubation was continued at 37%. Portions (0.1 ml.) were removed at intervals, diluted to 1 ml. with binding buffer and filtered to determine the amount of eH-labeled T7 DNA in RNA polymerase-T7 DNA complex (Hinkle & Chamberlin, 1972a). In a control reaotion to which no unlabeled T7 DNA was added, there was no decay of the RNA polymerase-3H-labeled T7 DNA complex in 3 hr. -O-O-, Incubation without rifampicin; --u-m-, incubation with rifampioin.

I Oo40

I 1 20 40

1 60

1 i SO 100 I

0

Time (set)

FIG. 2. Kinetioe of inactivation of RNA polymeraee by rifampicin. At zero time 0.7 a of RNA polymerase holoenxyme (fraction 6, 16,000 d(A-T) units/me;) WBBadded to 0.7 ml. (final volume) of binding buffer plus 1 mg BSA/ml. with or without 27.6 n%r-rifampioin at 37°C. Portions (0.1 ml.) were removed at intervals and added to 60 pl. of binding buffer containing 10 nmoles of T7 DNA plus 60 nmoles each GTP, CTP, UTP and [&‘P]ATP (16,000 cts/min/nmole). Incubation wa8 continued for 10 min at 37°C and acid-insoluble radioaotivity was determined. One unit of RNA polymerase activity catalyzes the incorporation of AMP at a rate of 1 nmole/hr. -O-O-, Incubation without rifampioin; -m-m-, incubation with rifampicin.

triphosphates, RNA polymerase holoenzyme has a half-life of 135 seconds. This relatively rapid decay of activity is probably due ta thermal inactivation, and the kinetics obey a first-order relation with an apparent rate constant of kobs= 5.1 x 10m3 set-I, When rifampicin is added to the reaction, the rate of inactivation of the enzyme is increased. In the presence of 2.75 x lo-* M-rifampicin, the apparent rate constant for inactivation of RNA polymerase is 17.3 x 10 -3 set-l (t+ approx. 40 set).

INTERACTION

OF RIFAMPICIN

AND

RNA

POLYMERASE

213

Under these conditions, where rifampicin is present in a tenfold molar excess over RNA polymerase, the kinetics for inactivation of the enzyme by rifampicin are pseudo first-order. In other words, although the binding of rifampicin to RNA polymerase is a second-order reaction, under conditions where the concentration of unbound antibiotic can be considered constant during the reaction, kobs= k, x [rifampicin] where k,,, is the apparent rate constant for the binding of rifampioin to RNA polymer&se and k, is the true second-order rate constant for the reaction. In the absence of DNA, the second-order rate constant for the inactivation of RNA polymerase by rifampicin can be calculated to be:

k2= (1’*3- 5.1)x 1o-3w-l = 4*4Xlo5M-1set-l 2+75x 1O-8 M (c) Kinetics of inactivation

of RNA polynaeraae holuenzyme-T7 DNA by rifampicin

complexes

The kinetics of inactivation of RNA polymer&se holoenzym*T7 DNA complexes at three different rifampicin concentrations are shown in Figure 3. Control experiments show that in the absence of rifampicin no loss of enzyme activity is observed for at least ten minutes at 37°C; that is, the binding of RNA polymerase to DNA completely protects the enzyme from thermal inactivation during the assay period. The period of T7 RNA synthesis was limited to 90 seconds. This minimizes the possibility of termination and reinitiation of T7 RNA chains (Chamberlin & Ring, 1972) ; under these conditions, the amount of RNA formed should be a direct measure of the amount of active RNA polymerase bound in DNA complexes. The number of complexes at t = 0 is experimentally defined as the amount of RNA synthesis obtained in 90 seconds when both rifampicin and the four ribonucleoside triphosphates are added to the complexes at the same time. At rifampicin concentrations below 10 H/ml., this value differs from that obtained in the absence of rifampicin by no more than 20% for RNA polymerase holoenzymet. The data indicste that RNA polymerase holoenzyme-T7 DNA complexes are much less susceptible to inactivation by rifampicin than free enzyme. For example, in the presence of 3.03 x 10es M-rifampicin, a concentration 100 times greater than that used in the experiment shown in Figure 2, the time required to in&&iv&e half of the initial complexes is 84 seconds. To determine the rate constant for the inactivation of the RNA polymerase holoenzyme-T7 DNA complex, the apparent first-order rate constant is plotted against the concentration of rifampicin (Fig. 4). For s, true pseudo firstorder reaction, a linear relation is expected between kobs and the rifampicin concentretion, with a slope equal to k,, the second-order rate constant for the reaction. While a linear relation is observed, extrapolation of the line to kobs= 0 yields a rifampicin concentration of 4 x 10S7 M. For a simple second-order reaction, kobs t In experiments where rifempicin is added to the holoenzym+T7 DNA aomplex together with nucleoside triphosphates, 8 low snd somewhat variable amount (10 to 20%) of aotivity is lost at rifampicin concentrations below 1 pg/ml. At last some of this “highly sensitive” fraction is due to synthesis by the core polymerase present in our preparations of holoenzyme (fraction 6 enzyme contains only about 0.8 equivalent of sigma), since it can be eliminated by addition of excese eigma (Mange1 & Chemberlin, unpublished observations). It seems probeble th8t this core polymerase is dependent for initiation on sigma subunit released by holoenzyme during the reaction. Binding of sigma to core polymeree+DNA complexes repidly reconstitutes holoenzyme but kale to rele8se of the enzyme from DNA before chain initiation (Hinkle & Chemberlin, 1972a); henoe, synthesis by this reconstituted enzyme is expected to be highly sensitive to rifampioin.

214

D. C. HINKLE,

W. F. MANGEL

AND

M. J. CHAMBERLIN

1°0W Time (ml

Fm. 3. Kinetics of immtivation of RNA polymerase holoenzyme+T7 DNA complexes by rifampi& at 37°C. Complexes were formed by adding 0.46 8g of RNA polymerase holoenzyme (frsotion 6, 24,000 d(A-T) units/mg) in 10 ~1. of RNA polymerase diluent (see Materials end Methods) to solutions containing 30 8l. of binding buffer 8nd 10 81. of 2.6 mm-T7 DNA. After 3 min et 37”C, 30 81. of binding buffer, with or without rifampioin, was added. Inoubation was oontinued for the indicated time intervals, then RNA synthesis was initiated by the addition of 20-d. samples containing 10 4. of binding buffer and 40 mnoles eaoh of GTP, CTP, UTP, and [%]ATP. The reaction was terminated 90 seo later and the acid-insoluble radio8otivity was determined. When rifempioin was added to the reaction together with the nuoleoside triphosphates, 100 pmoles of RNA were synthesized (100%). -@-a--, Incubation in 1.6 8~rifampioin; --O-O-, inoubetion in 6.1 @f-rifampicin. incubation in 3.0 8rd-rifampioin; - A-A-,

should be 0 at a rifampicin concentration of 0. We interpret this small deviation as due to oomplexing of a small but constant amount of rifampicin by some other component in the reaction, possibly the BSA added to protect the enzyme during dilution. Serum albumin is known to bind a wide variety of dyes and ionic compounds (Klotz, 1962). If this minor deviation is excepted, the data are consistent with a second-order reaction and give a value of k, = 3-5 x lo3 Me1 see-I, which is approximately two orders of magnitude smaller than that measured for free enzyme. When a similar experiment is performed with RNA polymerase holoenzyme-T7 DNA complexes at 15”C, comparable results are obtained (Fig. 5). The second-order rate constant is determined as before (Fig. 4) and is found to be approximately half that measured at 37°C (k, = 2.1 x lo3 116-l set-l). In each of these cases, the apparent rate constant for the inactivation of the holoenzyme-T7 DNA complex by rifampicin is many orders of mqnitude greater than that found for dissociation of the highly stable RNA polymerase-T7 DNA complex (EIinkle & Chamberlin, 1972a). Furthermore, the first-order rate constant for dissociation of the RNA polymerase-T7 DNA complex is over lOO-fold greater at 15°C than at 37°C. This suggests that the effect of temperature on the rate of inactivation of the holoenzyme-DNA complex by rifampicin is an inherent property of the rifampioin-RNA polymerase interaction end is not affected by the alteration of the stability of the holoenzyme-DNA complex et lower temperatures. (d) Kinetic% of inactivation of core polymerme-DNA

cmplexe~~ by r(fampicin

It has been suggested that the binary complex formed by the core polymer&se is more sensitive to attsok by rifampicin than that formed by the holoenzyme (Bautz & Bautz, 197Oob). Our results lead us to conclude that the drug does not affect the

INTERACTION

OF RIFAMPICIN r

I

AND

I

I

1

RNA 1

POLYMERASE

215

1

,

7 Rifompicin

COrWl

(,uM)

Fro. 4. Determination of the second-order rate oonstant for rifampioin stteck. Apparent &etorder rat~ oonstanta were caloulated using the data from Figs 3, 6 and 0 and are plotted ma a funotion of the rifempioin ooncentration in each reaction. --I)--@-, Holoenzpe-T7 DNA -, oore polymerase-T7 DNA oomplexes et 37°C; -O-O--, complexes at 37%; --Cl-m holoenzyme-T7 DNA oomplexes at 16°C.

Time

(mid

Fro. 6. Kinetics of inn&iv&ion of RNA polymerese holoenzyme-T7 DNA complex by rifampioin at WC. F&a&ion oonditions and procedures were the same as in Fig. 3, except that all inoubatione were performed at 16°C. When rifampioin wae added to the aaaay together with nuoleoside triphosphates 20 pmoles of RNA were synthesized (100%). --e-O-, Inoubetion in 1.5 einoubation in 3.0 F-rifampicin; rifampioin; -O-O-, -A---A--, inoubation in 6.1 p&trifampioin.

stability of the site-specific binding of RNA polymerase holoenzyme to T7 DNA, nor does the large alteration in the stability of the complex, caused by lowering the temperature to WC, dramatically alter the rate of attaok by rifampicin on the complex. Consequently, the notion that the core polymeraae-DNA complex ww attacked muoh more rapidly by rifampicin seemed puzzling. In agreement with Bautz & Bautz (1970), we find that the addition of rifampicin to the RNA polymerase-DNA complex

216

D. C. HINKLE,

W. F. MANQEL

AND

TILE

M. J. CHAMBERLIN

2

Effect of assay tim.e on inhibition of RNA polymercue by rifampicin Aasay time w4 RNA polymerese core polymemse

holoenzyme

Inhibition

2 10 2 10

by rif8mpicin (%I 0 26 60 96

Formation of RNA polymems+T’i DNA complexes w8a 8s described in the legend to Fig. 3. Complexes were formed with 100 nmoles of T7 DNA and 4.6 or 12.6 pg of holoenzyme (frection 6) or core polymerase (fraction 7B), respectively. After 10 min at 37”C, RNA synthesis w&s initiated with nucleoside triphosphates containing [a3aP]ATP and rifampicin (0.2 pg) where indioeted. RNA synthesis w8s terminsted efter 2 or 10 min as indicated. In 2-min assays in the absence of rifampicin, RNA polymer8se holoenzyme end core polymerase incorporated 408 and 69 pmoles of [GaPlAMP (loo%), respectively.

at the same time as nucleoside triphosphates in a standard RNA polymerase assay (10 min incubation) leads to extensive inactivation of core polymerase but has little effect on RNA polymerase holoenzyme (Table 2). However, the use of rate of inactivation of enzymic activity as a measure of the rate of attack by rifampicin has an important drawback. Since the enzyme-DNA complex is itself sensitive to attack by rifampicin, the amount of complex which is protected depends on the rate of RNA chain initiation. If this latter rate is slow relative to the rate of attack by rifampicin, then the amount of loss of RNA polymerase activity will not reflect the rate of attack by rifampicin on the complex. Thus, we must consider two competing reactions. (1) Binding of rifampicin

C-+ R- ‘*

CR

(2) RNA chain initiation k*

C + nNTP-C* where C represents the RNA polymerase-DNA complex, CR represents the enzymically inactive rifampicin adduct, C* represents the rifampicin-insensitive growing RNA polymerase-DNA-RNA complex, k, is the second-order rate constant for rifampicin attack and k* is an apparent rate constant for RNA chain initiation under standard assay conditions. Since the holoenzyme-DNA complex is attacked by rifampicin at a concentration of 6.09 x 10Vs M with a half-time of about 30 seconds, chain initiation by the holoenzyme must be complete in a much shorter time. However, the rate of RNA chain initiation by core polymerase is significantly slower at most initiation sites. This can be demonstrated directly by measurement of the rate of RNA chain initiation by core polymerase using y-labeled nucleoside triphosphates (Chamberlin & Ring, 1972). When the length of the incubation used in the standard assay for RNA polymerase activity is reduced, the fraction of core polymerase activity resistant to rifampioin is greatly increased (Table 2). Under these assay conditions, measurement of the rate of inactivation of the core polymerase by rifampicin (Fig. 6)

INTERACTION

OF

RIFAMPICIN

AND

RNA

POLYMERASE

217

Tune (mid

FIG. 6. Kinetioe of inactivation of oore polymemse-T7 DNA oomplexes by rifampioin at 37°C. The reeation conditiona and procedures were the seme 8s in Fig. 3, except thet the oomplex wat3 formed by adding 1.76 ILL of core polymeraee (fraction 7B, 13,000 d(A-T) units/mg) in 20 ~1. of RNA polymer8se diluent to solutions containing 10 d. of binding buffer end 20 4. of 2.4 mM-T7 DNA. When rifempioiu wae added to the raotion together with the nuoleoside triphosphates 36 pmoles of RNA wes synthesized (100%). This oorreaponda to about 60% of the amount of synthesia obteined in the absenoe of rif8mpioin. -a-a---, Incubation in 1.6 @%-rifampioin; -O-O-, inoubation in 3.0 q-rifampioin.

ahows that the intrinsic rate of attack by rifampicin on the core polymerase-DNA complex is comparable to the rate of attack on the holoenzyme DNA complex. Again, when the data are plotted as kobaagainst rifampicin concentration, a secondorder rate constant can be calculated (Fig. 4) and this is found to be 3.5 x lo3 M-~ set- l, the same as that obtained for binding of rifampicin to the holoenzyme-T7 DNA complex at 37°C. We conclude that the rate of attack of rifampicin on E. coli RNA polymerase is reduced but not eliminated when the enzyme binds to DNA. With T7 DNA, neither the site of binding on DNA nor the stability of the complex, nor the presence of sigma subunit greatly alters the rate of attack. The greater sensitivity of RNA synthesis by core polymerase in a standard RNA polymerase assay is chiefly due to a much slower rate of RNA chain initiation by enzyme lacking sigma subunit.

4. Discussion Early studies of the action of rifampicin on RNA polymerase indicated that it did not prevent binding of the enzyme to DNA (di Mauro et al., 1969; Umezawa et al., 1968). Wu $ Goldthwait (1969) showed that rifampicin blocks the binding of purine nucleoside triphosphates to the enzyme at a site which is probably responsible for the binding of the initial purine nucleoside triphosphate, which forms the 5’-terminus of the nascent RNA chain. This suggested that rifampicin blocked a step in RNA chain initiation. However, the observation of Sippel & Hartman (1970) that the binary complex between RNA polymerase and DNA was relatively protected from attack by rifampi&r, opened the possibility that rifampicin blocked a step in site selection. Bautz & Bautz (197Oa,b) studied the effect of rifampicin on T7 DNA aynthesis and concluded that the “enzyme-promoter” complex and only that complex was resistant to attack by rifampicin. The loss of activity by such a complex in the presence of rifampicin

218

D.

C. HINKLE,

W.

F. MANGEL

AND

M. J. CHAMBERLIN

was attributed to the rapid dissociation of the enzyme from DNA. They concluded that the core polymerase, and hence presumably also RNA polymerase holoenzyme bound at non-specific initiation points, was unable to form a rifampicin-resistant complex. Hence, they interpreted the amount of incorporation of y-labeled nucleoside triphosphate in the presence of rifampicin as a direct measure of the number of promoter sites for “early phage mRNA” on various phage DNA’s. In contrast, Sippel & Hartman (1970) found that both RNA polymerase holoenzyme and core polymerase formed resistant complexes, although the amount of complex resistant to rifampicin was lower when core polymerase was used. This complex was attacked at a much slower rate than that formed by the RNA polymerase holoenzyme. In the extreme case, the complex between RNA polymerase (either core or holoenzyme) and poly [d(A-T)] remains active for many minutes in the presence of rifampicin (Krakow, 1970, personal communication; M. Chamberlin, unpublished results). We have shown that the complex between RNA polymerase holoenzyme and T7 DNA dissociates at an extremely slow rate, which is many orders of magnitude slower than the rate of attack by rifampicin on this complex (Hinkle & Chamberlin, 1970). Furthermore, neither attack by rifampicin nor previous complexing of the enzyme to rifampicin affects the rate of dissociation of this complex. The rate of attack by rifampicin on the holoenzymeT7 DNA complex shows second-order kinetics with a rate constant approximately 100 times lower than that for attack of rifampicin on free enzyme. While the rate of dissociation of RNA polymerase holoenzyme from its promoter complex is far more rapid at 15°C than at 37°C the rate of attack by rifampicin is increased at the higher temperature. Our results suggest a model for the attack of RNA polymerase-T7 DNA complexes, in whioh the rate of attack is greatly reduced through binding to DNA. In its simplest form, the model postulates that the rate of attack by rifampicin is not strikingly altered by changes in the stability of the complex, by loss of sigma subunit from the complex nor by the site on DNA at which the enzyme is attached. The data do not rule out the possibility that RNA polymerase bound to DNA can exist in two or more states, which differ in their intrinsic rate of attack by rifampicin. For example, it has been suggested that there may be a conformation of the RNA polymerase holoenzym+promoter complex which is not attscked by rifampicin (Travers, 1971; Burgess, 1971). However, in the latter instance our results require that the equilibrium between sensitive and resistant states be rather rapid, since in experiments such as that shown in Figure 3, all of the RNA polymerase bound to DNA can be inactivated by rifampicin in a short time. If the complex between RNA polymerase holoenzyme and T7 DNA is, in fact, sensitive to attack by rifampicin, how can the relative insensitivity of RNA synthesis to inhibition by rifampicin, when the antibiotic is added with the nucleoside triphosphates, be accounted for? The simplest explanation is that the rifampicinsensitive bindary complex is rapidly converted through chain initiation into a ternary complex, which is completely resistant to the drug. So & Downey (1970) have shown that formation of the second phosphodiester bond in the nascent RNA chain is sufficient to produce a rifampicin-resistant complex. This provides an extremely convenient method for estimating the rate of chain initiation by the RNA polymerase haloenzyme-T7 DNA complex, and studies using this procedure are now in progress. The rate of attack of rifampicin on core polymerase appears to be reduced by binding to DNA to a similar extent as that of holoenzyme. However, the actual amount

INTERACTION

OF RIFAMPICIN

AND

RNA

POLYMERASE

219

of RNA synthesis in a ten-minute assay is greatly reduced for core polymerase when rifampioin is added with the nucleoside triphosphates. This difference can be decreased by carrying out assays of the rate of synthesis for very short times. We conclude that the greater sensitivity of RNA synthesis by core polymerase to rifampicin inhibition is due, in large part, to a much slower rate of chain initiation by core polymerase for most RNA chains. Since core polymerase probably initiates chains primarily at breaks in the DNA and other adventitious “false” initiation points, this lower rate of chain initiation is not surprising. Thus, while the conclusion (Bautz & Bautz, 1970a,b) that there is an intrinsic difference in the rate of rifampicm attack on core polymerase and holoenzyme DNA complexes or on “specific” as compared to non-specific complexes is probably not correct, the use of rifampicin may well allow such a discrimination to be imposed, since these different complexes may differ greatly in their rate of chain initiation. Hence, rifampicin promises to be an extremely useful tool for studies on the kinetics and sequence of events involved in RNA chain initiation. The meaning of the extreme resistance of some enzyme-DNA complexes to rifampicin has yet to be explained. Complexes such as those formed with poly[d(A-T)] and between core polymerase and T2 DNA (Sippel & Hartman, 1970) are far more stable to attack by rifampicin than the T7 promoter complex and are, in fact, more stable than our estimates of the rate of dissociation of core polymeraseT7 DNA complexes (Hinkle & Chamberlin, 1972a). This suggests that the resistant complex has a very different structure from that of the holoenzyme-DNA complex or the normal core polymerase-DNA complex. Such a resistant complex might result from the binding of RNA polymerase to breaks in the DNA. For example, binding at such a break might allow the 5’-phosphoryl terminus of the broken strand to mask the purine nucleotide (initiation) binding site, producing a highly resistant complex. Since such breaks are rare in our T7 DNA, this would explain our failure to observe such a resistant complex. Regardless of the exact structural features which assure complete resistance of these complexes to rifampicin, it is predicted from equilibrium considerations that the RNA polymerase-rifampicin adduct should be greatly reduced in its ability to bind in such a complex. We are grateful to Dr Jack Kirsch for his assistance in deriving the equation for inactivation of RNA polymerase complexes during RNA chain initiation. This investigation was supported by Public Health Service research grant no. GM12010 from the National Institute of General Medical Sciences; grant no. AI01267 from the National Institute of Allergy and Infectious Diseases; training grant no. CA05028 from the National Cancer Institute; and one of us (D. C. H.) was the recipient of U.S. Public Health Service Fellowship no. GM40468 from the National Institute of General Medical Sciences. REFERENCES Bautz, E. & Bautz, F. (1970a). Natzlre, 226, 1219. Bautz, E. & Bautz, F. (19708). CoZd Sp. Harb. Symp. Qpuznt. BioZ. 85,227. Berg, D., Barrett, K. & Chamberlin, M. (1971). Methods in Enzyrnclogy, 21, 506. Burgess, R. (1971). Ann. Reu. Biochem. 40, 711. Chamberlin, M. t Ring, J. R. (1972). J. Mol. BioZ. 70, 221. Hinkle, D. C. (1971). Ph.D. thesis, University of California, Berkeley, California. Hinkle, D. C. & Chamberlin, M. (1970). Cold Spr. Harb. Symp. Qwnt. B&Z. 85. 66. Hinkle, D. C. & Chamberlin, M. (1972a). J. Mol. BioZ. 70, 167. Hinkle, D. C. & Chamberlin, M. (1972b). J. Mol. BioZ. 70, 187.

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