Biochem. J. (1966) 100, 50

The Inhibition of Ribonucleic Acid Polymerase by Acridines

BY B. H. NICHOLSON* AND A. R. PEACOCKENuffield Department of Clinical Biochemistry, University of Oxford

(Received 19 October 1965)

1. The aminoacridines, proflavine (3,6-diaminoacridine) and 9-aminoacridine,and a hydrogenated derivative, 9-amino-1,2,3,4-tetrahydroacridine, were shownto inhibit in vitro the DNA-primed RNA polymerase of Escherichia coli. Theinhibition is strong with both proflavine and 9-aminoacridine, but weak with9-amino-1,2,3,4-tetrahydroacridine. 2. The extent to which the three acridines bindto calf-thymus DNA in the enzyme medium was studied spectrophotometrically.The extent of binding decreases in the order: proflavine, 9-aminoacridine, 9-amino-1,2,3,4-tetrahydroacridine. Some evidence was also obtained for interactionbetween the nucleoside triphosphate substrates and proflavine or 9-aminoacridine;no such interaction was detectable with 9-amino-1,2,3,4-tetrahydroacridine.3. Although the amount of acridine bound to DNA increases with increasing inhibi-tion, a stage is reached where an increase in acridine concentration still causes an

increase in inhibition, with practically no increase in the amount bound to DNA.4. Plots of reciprocal rates against the reciprocal ofDNA concentration were linearand had a common intercept when proflavine or 9-aminoacridine was present.Similar relations were obtained when the reciprocal concentration of nucleosidetriphosphates was plotted. The observations are interpreted kinetically in terms ofa competitive inhibition of the enzyme by proflavine or 9-aminoacridine and of a

kinetic role for the DNA analogous to 'activation'. 5. This suggests that inhibitoryacridine molecules can occupy the sites on the RNA polymerase that are specific forbinding the nucleoside triphosphate substrate or the bases of the DNA, when thesebecome accessible during the copying process.

Inhibition of the DNA-primed RNA polymerase(nucleoside triphosphate-RNA nucleotidyltrans-ferase, EC 2.7.7.6) has been observed botu in vitroand in vivo with several compounds (Goldberg &Rabinowitz, 1962; Waring, 1964a; Hurwitz, Furth,Malamy & Alexander, 1962). Inhibition byactinomycin D has been shown to be the result of aspecific combination with guanine residues in theDNA primer and the action appears to be the samein vitro and in vivo (Goldberg & Rabinowitz, 1962;Hamilton, Fuller & Reich, 1963). Other inhibitors,e.g. ethidium bromide (Waring, 1964b) and atebrin(Hochster & Chang, 1963), appear to be less specific,having no particular affinity for a base in the DNA.Their degree of inhibition, however, correlates wellwith their strong binding to DNA. The effect ofproflavine (3,6-diaminoacridine) on the activityin vitro ofRNA polymerase from E8cherichia coli Bhas been reported briefly (Waring, 1964b; Goldberg& Rabinowitz, 1962; Goldberg, Rabinowitz &Reich, 1962). Hurwitz et al. (1962) found that theinhibitory activity of proflavine is not restricted tothe RNA polymerase of E. coli and that, for a given

* Present address: Department of PhysiologicalChemistry, University of Reading, Berks.

proflavine concentration, the inhibition is greaterwith the DNA-primed DNA polymerase (EC2.7.7.7). From these results it has been inferred thatinhibition of RNA synthesis is caused by theattachment of proflavine to the primer DNA, whichwould be consistent with the established strongaffinity of the aminoacridines for nucleic acids(Peacocke & Skerrett, 1956). Nevertheless, otherenzymes, not involving DNA, are also known to beinhibited by acridines, e.g. urease and hexokinase(Albert, 1951, p. 306).The following describes a study of the action on

E. coli RNA polymerase of the aminoacridines,proflavine and 9-aminoacridine, and of a hydro-genated derivative of the latter, 9-amino-1,2,3,4-tetrahydroacridine; the relation of the inhibitionto the extent of binding was particularly sought.The results, though still allowing that inhibitionmay be partly due to binding of acridines by DNA,indicate that other mechanisms are involved.

MATERIALS AND METHODSMaterial&. E. coli B (500g. wet wt.) was supplied by the

Microbiological Research Establishment, Porton, Wilts.Cells were grown in continuous culture in a 201. culture

50

ACRIDINE INHIBITION OF RNA POLYMERASEvessel with a dilution rate of 0 75hr.1l, giving a meangeneration time of 093hr. at 37°. The culture mediumcontained: yeast extract, 10g.; glycerol, 10ml.; K2HPO4,22g.; KH2PO4, 17g.; tap water to 11., giving approx.pH6-8. Cells (2.7g. dry wt./l.) were harvested by centri-fugation and washed at 50 with buffer (10mM-tris-HCl-10mM-magnesium acetate, pH7-4). The suspension wasrecentrifuged, and the resulting slurry was frozen andstored at -20°.The DNA-dependent RNA polymerase was prepared by

the method of Chamberlin & Berg (1962). Their procedurewas followed through the streptomycin and protamineprecipitations, and the (NH4)2SO4 fractionations. Thechromatography on DEAE-cellulose was not found to benecessary and the enzyme pellet from the final (NH4)2SO4precipitation was dissolved in 2*8ml. of storage medium[0.1 M-(NH4)2SO4-50mM-tris-HCl (pH8-1)-0.1 mM-EDTA-5mM-glutathione-I mM-MgCl2] and stored in 5ml. glassvials in 0*4ml. portions at -70°. The activity of the RNApolymerase was followed during the purification by usingthe assay given below [with the addition of sufficientexogenous DNA (50,ug.) to saturate the priming require-ment of the enzyme]. For all assays, other than duringisolation, the enzyme was diluted by the addition of 10mm-tris-HCl (pH7X9)-10mm-MgCl2-10mM--l-mercaptoethanol-0-1mM EDTA containing bovine serum albumin (1 mg./ml.).DNA was prepared from fresh frozen calf thymus by the

procedure of Kay, Simmons & Dounce (1952) and extractedtwice by the chloroform-gel method (Gulland, Jordan &Threlfall, 1947). Unless otherwise stated, concentrations ofDNA are based on E260 values and expressed in terms of themolarity of DNA deoxyribonucleotides. A standardsolution of DNA (1mg. of DNA/I ml. has E1,.20) in 0.9%NaCl-10mM-sodium citrate was prepared at 00. ATP, CTPand GTP were obtained from L. Light and Co. Ltd., Coln-brook, Bucks., UTP (grade Sigma), yeast RNA (commercialgrade) was from the Sigma Chemical Co., St Louis, Mo.,U.S.A., and [8-14C]ATP (disodium salt) was from SchwarzBioResearch Inc., Orangeburg, N.Y., U.S.A. Membranefilters (type DA, porosity 0.65,u) were obtained from theMillipore Corp., Bedford, Mass., U.S.A., and similar 0 5-1.0, porosity filters from Oxoid Division, Oxo Ltd.,London, E.C.4.

Proflavine hemisulphate (British Drug Houses Ltd.,Poole, Dorset) was converted into the free base withaq. NH3. An 8g. portion was redissolved in 3200ml. ofboiling distilled water and filtered. The crystals formed oncooling were reprecipitated from 400ml. of 95% (v/v)ethanol and dried at 110°. The m.p. (sealed) was 2820.Approx. 6g. of 9-aminoacridine hydrochloride fromBritish Drug Houses Ltd. was dissolved in 275 ml. ofboiling water, and brought to pH6 with KOH; a slightprecipitate was removed by filtration. The free base wasprecipitated by addition of KOH to pH 11, filtered off andwashed successively with 0-1N-KOH, water and 15 ml. of95% (v/v) ethanol. Afterbeingdriedat 1100, itwas dissolvedin 200ml. of acetone. The solution was evaporated to 40 ml.and left to crystallize. The yellow crystals were dried at1100 (m.p. 2430). 9-Amino-1,2,3,4-tetrahydroacridinehydrochloride from the Aldrich Chemical Co., Milwaukee,Wis., U.S.A., had m.p. 2850 and was used as supplied.Solutions of acridines were prepared by dissolving weighedquantities in water with the addition of HCI to maintainneutrality. Concentrations were checked either spectro-

photometrically or volumetrically by the method of Bolliger(1940). The solutions were prepared as soon as possiblebefore use and kept in the dark.

Pancreatic ribonuclease was obtained from the SigmaChemical Co. The T1 ribonuclease was prepared fromTaka-diastase powder by the method of Rushizky & Sober(1962). Specific activity was determined by incubating0-8ml. of a solution of 7-5mg. of yeast RNA/ml. in 125mm-tris-HCl buffer, pH7-3, with 0-2ml. of enzyme solution for1 hr. at 37°. Then 1 ml. of ice-cold 5% (w/v) trichloroaceticacid-0-5% (w/v) La2(SO4)3 was added to the chilledreaction mixture, and the extinction was measured of asample of the supernatant made up to 5ml. with lOmN-HCI. The presence ofMg2+ or Mn2+ had no discernible effecton ribonuclease activity at the concentrations employed inthe assays of RNA polymerase.

Bovine serum albumin (fraction V; Armour Pharma-ceutical Co. Ltd., Eastbourne, Sussex) was used for enzymedilutions and as a standard for protein estimations, whichwere made by the method of Lowry, Rosebrough, Farr &Randall (1951).Procedure for the a8say of RNA-polymerase activity.

RNA-polymerase activity was determined by measuringthe amount of radioactive precursor rendered acid-insoluble. The incubation mixture contained, in 0-25ml.:tris-HCl buffer, pH8-0, 10,vmoles; MgCl2, ltmole;MnCl2, 0 25,umole; CTP, UTP and GTP, each 0 1,tmole;[8-14C]ATP, 0-1,mole and 50000 counts/min.; ,B-mercapto-ethanol, 3-0,umoles; DNA, 0-50kug.; enzyme, 11units(1 unit is the amount ofenzyme that catalyses the incorpora-tion of 1mumole ofAMP/lOmin. in the presence of 30,ug. ofDNA under the conditions of assay). When required, theacridine was added to the combined salt and nucleosidetriphosphate solutions before the DNA and enzyme. Theacridine solutions were prepared at various concentrationswhich, when added as a 0-05ml. portion, gave the desiredfinal concentration in 0*25 ml. In control experiments,0-05ml. of distilled water replaced the acridine solution.The reaction mixtures were incubated for 10min. in 10ml.conical centrifuge tubes held in a water bath at 37°.

After incubation the tubes were transferred to a boiling.water bath; the inclusion of this denaturation step, recom-mended by Waring (1964b), led to a more finely dividedprecipitate and better reproducibility of results. After3 min. the tubes were placed in an ice-water bath for afurther 5min. Then 0.1 ml. of yeast RNA (2mg./ml.) wasadded as a co-precipitant followed by 0-35ml. of ice-cold10% (w/v) trichloroacetic acid. After 15min. at 0° thesuspension was applied to 13mm.-diam. membrane filtersand filtered under vacuum. The filters were washed withfive successive 3ml. portions of 5% (w/v) trichloroaceticacid and finally with 2ml. of 95% (v/v) ethanol. Thefilters were dried under an infrared lamp and mounted on15mm. planchets. Counting was effected in a windowlessgas-flow counter and was continued until the error waswithin + 3%. Incorporation in m,umoles was measured bydividing the counts so obtained by the specific activity ofthe precursor. All samples were assumed to be infinitelythin.

Acridine binding by DNA. Investigation ofthe binding ofacridines to DNA by the spectrophotometric method waspossible only over the concentration range where Beer's lawwas obeyed. Conditions were choseni that simulated theconditions in the enzyme assay as nearly as possible. Bovine

Vol. 100 51

B. H. NICHOLSON AND A. R. PEACOCKEserum albumin of the same concentration (w/v) replacedenzyme in the incubation mixture. A solution of acridine in2-5 ml. of this modified incubation medium was equilibratedat 37°. Small volumes ofDNA solution were added and thedecreases in extinction measured in a Unicam SP. 500spectrophotometer with a Gilford attachment. Measure-ments were taken at appropriate wavelengths where themaximum change in absorption occurred on the addition ofDNA to the acridine solution. These wavelengths were: forproflavine, 440m,; for 9-aminoacridine, 400m,u; for9-amino-1,2,3,4-tetrahydroacridine, 320mju. From thedecrease in extinction at these wavelengths when DNA wasadded, it was possible to calculate the no. (r) of acridinemolecules bound/nucleotide (Peacocke & Skerrett, 1956).

Acridine binding by nucleoside tripho8phates. Thepossibility of complex-formation between acridines andnucleoside triphosphates was investigated by using thespectrophotometric method. The spectra of the acridineand of the nucleoside triphosphate were compared withthat of the mixture of acridine and nucleoside triphosphateover the wavelength range 220-750m,u in a Perkin-Elmercontinuous-recording spectrophotometer (model 137 UV).The spectrum of proflavine (36ILM) in 40mM-tris-HCl

buffer, pH 8-0, was determined; 0-05 ml. of 10mM-ATP wasadded to 5ml. of the same proflavine solution, and thespectrum determined again. A 0-05ml. sample of 10mM-ATP was also diluted with 5ml. of buffer and the spectrumwas recorded on the same chart. The same procedure wasfollowed with excess of the other three nucleoside tri-phosphates, at a final concentration of 0.1 mm, and with theother two acridine derivatives, at final concentrations of36Mm for 9-amino-1,2,3,4-tetrahydroacridine and 69Mmfor 9-aminoacridine. All spectra were determined at roomtemperature.

RESULTS

Inhibition 8tudies. The effect of different con-centrations of DNA primer on the activity of theenzyme is shown in Fig. 1. From these results, theoptimum concentration was taken to be 30 rig. ofDNA/0-25ml. of assay mixture. At the enzymeconcentration normally employed (1 units), incor-poration up to 6000 counts/min. proceeded linearlyduring the lOmin. incubation, in the absence ofacridines. Hence the incorporation after 10min.was proportional, within the experimental limits, tothe initial rate of incorporation and so to the initialvelocity of RNA formation. The initial rate ofincorporation, with 30,ug. of DNA, was determinedin this way with different enzyme concentrationsbetween 0-1 and 16 units and was shown to beproportional to the enzyme concentration. Toobtain the main features of the kinetics, the highlyprobable assumption was made that the decreasedincorporation in the presence of acridine was like-wise linear for the first lOmin. under the sameexperimental conditions.

In the absence ofDNA primer, incorporation wasless than 120 counts/min. above the background of18 counts/min., which shows that the presence ofDNA increases the rate 50-100-fold. Table 1

12-5

10-0 - >

0

o 5 050

0 10 20 30 40 50

DNA (,ug.)

Fig. 1. Incorporation ofAMPinto acid-precipitable materialby 1 units of E. coli RNA polymerase in the presence ofdifferent amounts of calf-thymus DNA. The conditions ofincubation are given under 'Procedure for the assay ofRNA-polymerase activity' in the Materials and Methodssection.

Table 1. Incorporation of [8-14C]AMP into acid-precipitable material by the action of E. coli RNApolymerase

The conditions of incubation were as described under'Procedure for the assay of RNA-polymerase activity' inthe Materials and Methods section, with the further varia-tions indicated (incubation time was 10min.). In general,11 enzymes units were primed by 30,tg. of calf-thymusDNA.

Incubationtemp. Variations to conditions370 None37 1000 for 5min., 30min. at

370 with pancreaticribonuclease

37 100° for 5min., 30min. at370 with T1 ribonuclease

37 100° for 5min., 30min. at370 with both pancreaticand T1 ribonucleases

0 With proflavine (250ytM)37 No DNA primer

Incorporation(counts/min.)

5400660

1320

420

240120

presents some of the results of experiments under-taken to test whether the system had the expectedcharacteristics. In the experiments with ribo-nuclease, the amount of enzyme added was thatcalculated, on the basis of specific activity, to besufficient to hydrolyse the RNA formed by thepolymerase. It had previously been shown, by

52 1966

ACRIDINE INHIBITION OF RNA POLYMERASE

0 0 20 30 40 50

c (PM)

Fig. 3. Dependence of the no. (r) of acridine moleculesbound/DNA nucleotide on the free acridine concentration(c): 0, proflavine; I, 9-aminoacridine; *, 9-amino-1,2,3,4-tetrahydroacridine. Values were determined at 37° in themodified incubation medium by the spectrophotometricmethod described under 'Acridine binding by DNA' in theMaterials and Methods section.

150

20

90

0co460

20

0

-,

_-

0 PO i60 240

Concn. of inhibitor (/lM)

30

W-

320

Fig. 2. Extent of the inhibition by proflavine (o), 9-aminoacridine (LOI) and 9-amino-1,2,3,4-tetrahydroacridine(e) of the activity of 1lunits of E. coli RNA polymeraseprimed by 30,ug. of DNA. The conditions of incubation aregiven under 'Procedure for the assay of RNA-polymeraseactivity' in the Materials and Methods section.

reference experiments with polyadenylic acid as

substrate for the ribonuclease, that, under suchconditions, if all the [8-14C]AMP were incorporatedas polyadenylic acid, the amount split by the ribo-nuclease would be less than 0.1% (i.e. less than6 counts/min.).The effect of the addition of various amounts of

acridines to enzyme mixtures, containing a constantamount of DNA, is shown in Fig. 2. On a molarbasis, proflavine inhibited more than did 9-amino-acridine, whereas 9 - amino - 1,2,3,4 - tetrahydro-acridine inhibited very little. The 25% decreasein incorporation observed at a concentrationof 1OmM-9-amino-1,2,3,4-tetrahydroacridine was

observed with 9-aminoacridine at 35 pm.Binding 8tudies. The extent of binding of the

acridines with DNA in the assay medium is inagreement with previous work (Fig. 3): the bindingto DNA was shown to decrease with increase insalt concentrations (Peacocke & Skerrett, 1956;Drummond, Simpson-Gildemeister & Peacocke,1965), and intheplot ofragainst the concentration (c)offree unbound acridine therewas a plateauregionatr0 14, corresponding to the completion of strongbinding on calf-thymus DNA in 0 1M-sodiumchloride (pH6.9, 25°). In 60mi-sodium chloridethe plateau occurred at rO16. The type of ionpresent also had an effect on the binding: the addi-tion of Mg2+ (5mM-magnesium chloride) decreasedbinding by approx. 20% more than would beexpected from considerations of ionic strength alone(Peacocke & Skerrett, 1956). Thus the depressionofthe binding (Fig. 3) to r0-135 for proflavine in the

complex assay medium (10.05, 4nmi-magnesiumchloride) was in accord with observations withsimpler solvents. This resemblance at the lower r

values suggested that the binding curve for thisenzyme medium obtained by the spectrophoto-metric method might be extended to higher r values(greater than 0 2) by using previous resultsobtained by equilibrium-dialysis measurements inthis range of r (Fig. 9 in Peacocke & Skerrett, 1956).These results consist ofa complete binding curve forherring-sperm DNA (10.5mg. atoms ofphosphorus/1., Olm-sodium chloride, pH6*9, 200), which had a

plateau region at rO22. The binding curve deter-mined here for the enzyme assay medium was

extended by multiplying these r values found byequilibrium dialysis by the ratio of r at therespective plateau regions (0.135/0.22).

Since the binding curve (r against c) does not varysignificantly with changes in DNA concentration(TA, in g.atoms of nucleotide phosphorus/I.)(Peacocke & Skerrett, 1956), it was possible to findthe total acridine concentration (TL) correspondingto a particular r value at a given DNA concentrationby using the relation:

TL= TA.r+c

By substituting corresponding pairs of r and cvalues from Fig. 3, and with TA equal to that in theenzyme assay, a series of TL values was obtainedand plotted against r. Values of r could then beread from this graph for the particular totalproflavine concentrations (TL) used in the inhibition

80

60

0

-0I

40

Vol. 100 53

B. H. NICHOLSON AND A. R. PEACOCKE

100

° 800

'-4

0 60

.] 40C)

100

1-

2ci0

0'4-40

-4

4.C..;20 L _ I _I 1 I__

0 40 80 120 160 200TL (,UM), c (,UM) or 103r

Fig. 4. E. coli RNA-polymerase activity (control 100%) of11 units of enzyme primed by 30,utg. ofDNA in the presenceof proflavine, plotted against: o, fraction bound (r); o,total concentration (TL); *, free concentration (c) ofproflavine. The conditions of incubation are given under'Procedure for the assay of RNA-polymerase activity' inthe Materials and Methods section. Points below thehorizontal double lines were obtained by calculation fromprevious data (see the text).

50 -

i- _ * 1 _ -1 1

30 60 90 120 150

TL (MM), C (,UM) or 103r

Fig. 5. E. coli RNA-polymerase activity (control 100%) of11 units of enzyme primed by 30,tg. of DNA in the presenceof 9-aminoacridine, plotted against: E, fraction bound (r);0, total concentration (TL); *, free concentration (c) of9-aminoacridine. The conditions of incubation are givenunder 'Procedure for the assay ofRNA-polymerase activity'in the Materials and Methods section.

experiments. The corresponding free proflavineconcentration (c) could also be calculated bysubstitution in the above equation.

Relation of inhibition to the binding of the acridineson DNA. Fig. 4 shows the decrease in activity,expressed as a percentage of the control (noacridine), at different total proflavine concentra-tions (TL) when 30,ug. of DNA is used as primer in0-25ml., i.e. TA is 369tLM. After the activity hasdropped to 36% ofthe control, no further inhibitionwas observed, even at the highest proflavine con-centrations employed.

Fig. 4 also shows the variation of activity with rand with the concentration (c) of free proflavine.The curves above the horizontal double lines inFig. 4 are based on the experimental observationsand r values determined for the enzyme assaymedium; the curves below the double lines arederived from r values on the binding curve extendedby the procedure described above. The resultsobtained in similar experiments with only 5,ug. ofDNA as primer gave similar curves to those ofFig. 4, with inhibition reaching 80%. The corres-ponding results with 9-aminoacridine and 30,ug. ofDNA as primer are shown in Fig. 5. No equi-librium-dialysis measurements were available with9-aminoacridine, so that no r or c values are knownaccurately above the plateau region, where r is0-087 (Fig. 3). This limit is also indicated by thehorizontal double lines in Fig. 5.The binding curve of 9-amino-1,2,3,4-tetrahydro-

acridine was limited by the range available to thespeDtrophQtQmetric method, i.e. up to the plateau

region where r is 0 033, TAis 369 um and TLis 37-2/AM.The enzyme reaction mixture, which contained thesame concentration (TA) of DNA, showed nosignificant inhibition until TL was 150uM (Fig. 2)and, at TL as high as 1OmM, 74% of the controlactivity still remained.

Kineties of inhibition. In a further series ofexperiments the inhibition by the same concentra-tion of dye with various amounts of DNA primerwas investigated. The chosen concentrations ofacridine caused approx. 50% inhibition with 30,ug.of DNA primer (150,uM-9-aminoacridine and48ptM -proflavine). 9-Amino- 1,2,3,4 -tetrahydro-acridine was not tested because of its lack ofinhibitory activity at low concentrations andbecause of its limited solubility. The results fromthese experiments were expressed in the form of areciprocal plot, (incorporation of [8-14C]ATP)-lversus (DNA concentration)-l (Fig. 6). Theseresemble the usual Lineweaver & Burk plots (1934),although DNA in this system is not a true sub-strate.The results of similar experiments in which the

concentration ofnucleoside triphosphates was alonevaried are shown in Fig. 7; constant volumes ofdifferent dilutions of a combined stock solution ofall four nucleoside triphosphates were added to eachassay. At very low concentrations of the nucleosidetriphosphates, above 0 5 on the abscissa, there wasa tendency for the line to curve upwards, which mayhave been due to the large change in the ratio ofMg2+ to nucleoside triphosphates.

54 1966

ACRIDINE INHIBITION OF RNA POLYMERASE80

-0-5 0 0 5 1-0 5 2 0

10-4/[DNA] (M-1)

Fig. 6. Reciprocal plot of the activity (counts/lOOsec.) of11 units ofE. coli RNA polymerase and of the concentrationof DNA primer in the presence of acridines: 0, control;o 481 ,um-proflavine; O, 150,uM-9-aminoacridine. Theconditions of incubation are given under 'Procedure for theassay of RNA-polymerase activity' in the Materials andMethods section. Each point represents the mean of threevalues.

-5 0 5 l0 15

10-3/[Nucleoside triphosphates] (M-1)

Fig. 7. Reciprocal plot of the activity (counts/lOOsec.) of3*5 units ofE. coli RNA polymerase primed by 5,tg. ofDNAand of the concentration of the nucleoside triphosphatesin the presence of acridines: 0, control; o 50OM-proflavine;LO1, 50,uM-9-aminoacridine. The conditions of incubationare given under 'Procedure for the assay ofRNA-polymeraseactivity' in the Materials and Methods section. Each pointrepresents the mean of three values.

DISCUSSION

Several enzymes are known to be inhibited byacridines. For example, the antimalarial drugatebrin [6-chloro-9-(4-diethylamino-1-methylbutyl-amino)-2-methoxyacridine] inhibits cholinesterase

to the extent of 50% at 10DuM, and lactate de-hydrogenase is strongly inhibited at a concentrationof 1 0mM (Albert, 1951, p. 305). Many of theenzymes inhibited possess coenzymes that in excessprevented inhibition or even reversed it, if addedafter atebrin (e.g. D-amino reductase with FAD,cytochrome reductase with riboflavine, glucose6-phosphate dehydrogenase with NADP). Atebrininhibits RNA polymerase (Hochster & Chang, 1963)and Acridine Orange inhibits phosphorolysis ofpolyadenylic acid by polynucleotide phosphorylase(Beers, Hendley & Steiner, 1958). RNA polymerasein preparations from HeLa cells was inhibited byactinomycin (2,ug./ml.), proflavine (200,g./ml.)and acriflavine (200,tg./ml.) to the extents of 51, 35and 69% respectively, i.e. incorporation of nucleo-tides into RNA was decreased by this fraction(Goldberg & Rabinowitz, 1962). This inhibitioncould be completely reversed by adding denaturedDNA and partly reversed by adding native DNA.It was also shown that low concentrations of pro-flavine (1.5pM) depressed to half its original valuethe synthesis ofRNA inthis system, when primed bya copolymer of the deoxyribonucleotides of adenineand thymine (Goldberg et al. 1962). Hurwitz et al.(1962) compared the action of proflavine on RNApolymerase and DNA polymerase and showed thatproflavine (30Mm) decreased the activity of RNApolymerase by 30% and that of DNA polymeraseby 85%.The inhibitory activities towards E. coli RNA

polymerase of the three acridines used in thepresent study were in the order: proflavine - 9-aminoacridine>9 - amino - 1,2,3,4 - tetrahydro-acridine, which is parallel with their antibacterialactivity (Albert, 1951, pp. 251-263) and with theirdecreasing ability to bind to DNA (Fig. 3). Thisseems to implicate the binding of the acridine tothe DNA as the cause of the inhibition, as appearsto be the case with ethidium bromide (Waring,1964a). However, when the fraction of the originalenzyme activity is plotted (Fig. 4) against the no.(r) of proflavine molecules bound/DNA nucleotidein the enzyme system, the inhibition is seen toincrease not only with r up to a value corre-sponding to the plateau region of the DNA-binding curve (Fig. 3) but also beyond this point.That is, inhibition increases with proflavine con-centrations when r hardly changes at all. It istherefore possible to decrease the level of incorpora-tion of nucleotides into RNA in this system byincreasing the proflavine concentration beyond80tM, even though, over this range, very little ofthe extra proflavine is binding to the DNA. Thesame situation is observed with proflavine inhibitionof the same amount of enzyme at lower concentra-tions (5,ug.) of DNA primer and with 9-amino-acridine (Fig. 5). It is clear that inhibition at the

55Vol. 100)

B. H. NICHOLSON AND A. R. PEACOCKEhigher acridine concentrations cannot be caused bybinding to the DNA, although this undoubtedlyoccurs. Since the extension and partial unwindingof the DNA molecule caused by the binding ofacridines also reaches a maximum at the plateauvalue of r (Drummond, Pritchard, Simpson-Gildemeister & Peacocke, 1966), this extensioncannot be the cause of the inhibition of the RNApolymerase by the acridines, since it is constant inthe range of acridine concentrations over whichinhibition is still increasing. This behaviour ismanifest even if the decrease in activity is plottedas a function of total (TL) or of free (c) acridineconcentrations (middle and lower curves in Figs.4 and 5).When the reciprocal of the rate of incorporation

is plotted against the reciprocal of the total DNAconcentration, at constant nucleotide concentra-tion, a linear plot is obtained that has the sameintercept, with an enhanced slope, if proflavine or9-aminoacridine is present (Fig. 6). This is the typeof relation characteristic of competitive-inhibitionkinetics. When the reciprocal of the amount of'uncomplexed DNA' was plotted as the abscissaagainst the reciprocal of the rate (by 'uncomplexedDNA' is meant total DNA minus DNA inactivatedby acridine, on the basis of one nucleotideinactivated/acridine molecule bound) then straightlines were again obtained. These had almost thesame intercept on the ordinate but a decreased slope.Presumably, therefore, within the limits of theexperimental error of the kinetic experiments, theamount of 'uncomplexed DNA' is approximatelyproportional to the total DNA present, and so thislatter quantity has been used in the kinetic analysis.The corresponding plot of reciprocal rate against

reciprocal nucleotide concentration, at constantDNA concentration, was also linear and againexhibited the form characteristic of competitiveinhibition when proflavine or 9-aminoacridine waspresent (Fig. 7). These curves can be explained onthe following basis. In the absence ofinhibitor, bothDNA and the nucleotide triphosphates are essentialfor the reaction, but only the latter is the strictsubstrate and reacts to give the product, RNA.The role of DNA is therefore kineticallyanalogous to that of an activator and the equilibriamay be formulated as follows, where E is enzyme(RNA polymerase), S is substrate (nucleosidetriphosphates) and D is DNA primer:

E+S =ES; KJ = [E][S]/[ES] (1)

E+D =ED; Kd = [E][D]/[ED] (2)

ED + S = EDS; KJ = [ED] [S]/[EDS] (3)

Square brackets denote molar concentrations, [D]meaning the DNA concentrations in g.atoms ofphosphorus/l. and [E] the concentration of freeenzyme.The assumptions are made that: (i) the equilibria

(1)-(3) are established and not disturbed by theformation of product (i.e. Michaelis kinetics);(ii) KJ= KJ, i.e. the binding of substrate on enzymeis not affected by the binding of DNA; (iii) only thecomplex EDS can yield the product (RNA);(iv) the DNA is not affected by this last stage, i.e. theDNA is an 'activator' and at the end remainsunchanged. (The further equilibrium,

ES+D =EDS,

is redundant.) It is readily shown (Dixon & Webb,1964) that:

v = k[EDS] = k[E]o

IS]) [D]) (5)where [E]o is total concentration of enzyme.Eqn. (5) predicts the observed linearity, in theabsence of acridine, of the Lineweaver-Burk plotsof Figs. 6 and 7 for constant [S] and [D] respectively.As mentioned above, the kinetics of inhibition by

proflavine and 9-aminoacridine are of the competi-tive form, both for variable [D] at constant [S] andfor variable [5] at constant [D]. It is thereforeproposed that the inhibition occurs as a result oftheacridine (I) combining with the enzyme and that theenzyme-acridine complex (EI) cannot yield theactive form, EDS. Thus:

E+I -EI; K,= [E] [I]/[EI] (6)

and, more fully:

+1) ED -4+I

EI E EDS > product (RNA)

+b ES )

(7)

In this case:

S] [D] [S] [D] [I][E]o = ['E]Il+-+-----+ +K Kd K .Kd K4

whence:

v = k[EDS] = kK[S][D][E]and:

I 1 K K1+[KD]+Kd [I]

V=

rl ,I+ (8)

kl 1;D + product (RNA); If [D] is varied at constant [S] and constant [I],rate = v = k[EDS] (4) the plot ofv-1 versus [S]-1 is linear. As [I] inreases

56 1966

Vol. 100 ACRIDINE INHIBITION OF RNA POLYMERASE 57

from zero, the intercept is unaltered but the slopeshould increase, since eqn. (8) may be written:

K=fKd(l iS] K, Kd JI 1+( IS)

k[E]o Ki [S] j[D] k[E]o (9)

This is the behaviour observed (Fig. 6).If [S] is varied at constant [D], the plot of v-1

versus [S]-1 is also linear and, if [I] increases fromzero, the intercept is again constant (but now equalto (1 +Kd/[D])/k[E]o), and the slope should increase.This is as observed (Fig. 7).

Eqns. (8) and (9) therefore fit the observedphenomena and support the kinetic scheme shownin eqn. (7). The values of the various constantsdeduced from this analysis are: K. 2*2 x 10-4 moles/1.; Kd42 x 10-5 moles/l.; K, for proflavine 5.4 x 10-9moles/l.; K, for 9-aminoacridine 2-4 x 10-9 moles/l.The DNA bases are more strongly bound (K.IKd isapprox. 5) than the free nucleoside triphosphatebases and the dissociation constant of proflavinefrom the enzyme sites is twice that of 9-amino-acridine.The form of the kinetic relationship indicates

that molecules ofthe inhibitory acridine can occupysites on the enzyme to which either the DNA or thenucleoside triphosphates normally attach them-selves. The recognition process required to produceRNA related to the DNA in nucleotide sequenceimplies that the DNA is in some way opened out sothat its bases are close to the enzyme. The site onthe enzyme that binds the DNA therefore needsonly to be specific for the base moiety of thedeoxynucleotide, and so it is conceivable that thesimilarly sizedflat ring ofthe acridine could competefor both this site and that normally occupied by thenucleoside triphosphate or its base. This also makesit clear why the inhibitory activity of the variousacridines should be parallel to their ability to bindto DNA, since the same stereochemical factorswould influence the ability to interact with the baserings ofDNA and with the enzyme sites capable ofholding such rings. This parallelism therefore neednot be interpreted to mean that the binding toDNA is itself necessarily the cause of the inhibitoryaction on RNA synthesis. Although such bindingmay still make some contribution to the inhibition,it cannot explain the main features ofthe inhibition,in particular the competitive form of theLineweaver-Burk plots. The existence of a weakinteraction between the acridines and the nucleosidetriphosphates is also a possibility since the extinc-tion of a proflavine solution decreased when asolution ofnucleoside triphosphates was added to it,although there was no shift in the wavelength ofmaximum absorption.

The inability of the buckled molecule of 9-amino-1,2,3,4-tetrahydroacridine to inhibit RNA synthesisin the DNA-primed system must result from itsinability to attach to the enzyme sites whoseconfiguration normally sequesters the flat base ringsof either DNA or the nucleoside triphosphates.This lack of inhibitory power in the enzyme systemhas the same structural basis as its low affinity forDNA (Peacocke & Skerrett, 1956) and, presumably,its low bacteriostatic power (Albert, 1951, pp. 259-263).In a similar study of the inhibition of RNA

polymerase by ethidium bromide, Waring (1964a)found that complete inhibition could be achievedand that a plot of percentage of control activityagainst r gave the same straight line for differentexperimental conditions. This was consistent withthe view that the binding of ethidium bromide tothe DNA was the cause of inhibition, rather thaninterference with the binding ofDNA (or nucleosidetriphosphates) to the enzyme. However, even thiscorrelation does not entirely eliminate the latterpossibility, which appears from the foregoing to bethe likelier explanation of the characteristics ofinhibition by the acridines. This difference betweenethidium bromide and the acridines is unexpectedin view of the close similarity of their bindingcharacteristics to DNA (Waring, 1965). The causeof the difference is probably therefore to be soughtin the greater ease with which the acridines occupythe active sites on the enzyme, and this couldreasonably be attributed to the ethidium cationbeing larger in area than one heterocyclic base ringof either DNA or nucleoside triphosphate.

The authors acknowledge the help afforded by a ResearchFellowship of the Department of Scientific and IndustrialResearch (to B. H. N.), and the facilities provided byMr J. R. P. O'Brien, Director of the Nuffield Department ofClinical Biochemistry, University of Oxford. They wereparticularly helped by the co-operation of the Director andstaffofthe Microbiological Research Establishment, Porton,Wilts., in providing adequate amounts of E. coli cells forthe enzyme preparation.

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