JOURNAL OF BACTERIOLOGY, July, 1966Copyright © 1966 American Society for Microbiology

Vol. 92, No. IPrinted in U.S.A.

Tetracycline Inhibition of Cell-FreeProtein Synthesis

II. Effect of the Binding of Tetracycline to the Coinponents of the SystemL. E. DAY

Pfizer Medical Research Laboratories, Chas. Pfizer & Co., Inc., Groton, Connecticut

Received for publication 10 May 1966

ABSTRACTDAY, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of

cell-free protein synthesis. II. Effect of the binding of tetracycline to the componentsof the system. J. Bacteriol. 92:197-203. 1966.-When tetracycline, an inhibitor ofcell-free protein synthesis, was preincubated with each component of the Escher-ichia coli cell-free system, i.e., ribosomes, soluble ribonucleic acid (sRNA), poly-uridylic acid (poly U), and S-100 (supernatant enzymes), only the ribosomal-boundantibiotic was inhibitory to the cell-free assay. Experiments designed to furtherlocalize the site of inhibition to either the 50S (Svedberg) or the 30S ribosomal sub-unit were not conclusive. Tritiated tetracycline (7-H3-tetracycline) was bound toisolated 50S ribosomes, and these were recombined with 30S subunits to form 70Sribosomes. When these ribosomes were dissociated and the subunits reisolated, theantibiotic was found with both the 50S and the 30S particles. The same results wereobserved when the tetracycline was initially bound to the 30S subunit.

The previous report in this series (3) describedstudies which demonstrated the bindingof tetra-cycline to several of the various nucleic acidcomponents of the Escherichia coli cell-free pro-tein-synthesizing system. Although the antibioticwas observed to bind to polyuridylic acid (polyU) and polyadenylic acid (poly A), to solubleribonucleic acid (sRNA), and to both subunitsof 70S (Svedberg) ribosomes, no evidence wasgiven that indicated which, if any, of these bind-ing sites represented the primary locus of inhibi-tion of the cell-free system. Connamacher andMandel (2) suggested that the antibiotic in-hibits protein synthesis by binding directly to themessenger ribonucleic acid (mRNA) and pre-venting the attachment of sRNA to the mRNA-ribosome complex, whereas Suarez and Nathans(18) postulated that tetracycline binds to theribosomes, thus obstructing one of the twoaminoacyl-sRNA binding sites.

This report contains the results of experimentsdesigned to resolve the question of the localityof the site of inhibition in the cell-free system.This was approached by preincubating each ofthe components of cell-free assay with tetracy-cline, followed by extensive dialysis or centrifu-gation through 10% sucrose to remove unboundantibiotic, and utilization of the component inthe poly U-directed CI4-phenylalanine incorpora-

tion assay. The preincubation was carried outwith poly U, sRNA, supernatant enzyme frac-tion (S-100), and ribosomes. Only the ribosomal-bound antibiotic was inhibitory to the cell-freesystem. The antibiotic was then preincubatedwith isolated ribosomal subunits, and these wererecombined with untreated subunits to form 70Sribosomes with the tetracycline bound to eitherthe 50S or the 30S particle. This was done in aneffort to localize the site of inhibition to one orthe other subunit. It was observed that the tetra-cycline was equally as effective as an inhibitorof the cell-free system when bound to the 50Sor to the 30S portion of the 70S ribosome.Further experimentation, however, indicatedthat when 7-H3-tetracycline was initially boundto one species of isolated ribosomal subunits,subsequent manipulations with the other speciesto form 70S ribosomes resulted in the antibioticbeing associated with the both subunits. It is notpossible by this technique to resolve the locationof the site of inhibition on the ribosome.

MATERIALS AND METHODS

Culture and ctultural methods. E. coli W was main-tained and cultured as before (3).

Preparation of cell-free extracts, ribosomes, andhybrid ribosomes. The preparation of cell-free extracts,ribosomes, and ribosomal subunits has been described

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previously (3). Ribosomes [705 (TC)] were preparedby incubating 360 ,ug of tetracyline with 18 mg of70S ribosomes (ribosomal protein) in 0.01 M tris(hy-droxymethyl)aminomethane-HCl (Tris), pH 7.3, and0.01 M MgCl2 at 37 C for 15 min. This mixture wasthen layered over 10% sucrose in the Tris-HCl andMg+ buffer and was centrifuged at 5 X 104 rev/minfor 1.5 hr in the 50 rotor of the Spinco model L2-50at 3 C. The pellet was homogenized in 0.01 M Tris-HCl (pH 7.3) and 0.01 M MgCl2, and the centrifuga-tion through sucrose was repeated. Control ribosomeswere treated in the same manner, except that tetra-cycline was omitted from the initial reaction mixture.

Isolated 50S ribosomal subunits (19.5 mg of pro-tein) and 30S subunits (16 mg of protein) were incu-bated with 390 and 320 J,g of tetracycline, respectively,under the conditions described above. Both reactionmixtures were then dialyzed at 5 C against 0.01 MTris-HCl (pH 7.9), 0.01 M MgCI2, and 0.02 M NH4CIfor 4 hr, and against fresh buffer for 16 hr. The ribo-somal suspensions were then removed from the dialy-sis tubing, assayed for protein, and recombined witheach other and with control subunits to form the 70Sribosomes. Control subunits were treated in the samemanner as the tetracycline-treated subunits, exceptthat tetracycline was omitted from the initial reactionmixture. The subunits were recombined to form 705ribosomes by mixing 2 parts of 50S subunits (basedon protein) to 1 part of 305 subunit in the presenceof 0.01 M MgCI2. Sucrose density-gradient centrifuga-tion analysis indicated that the treated subunits re-combined to form typical 70S ribosomes. The recon-stituted ribosomes were designated as follows:50S(TC)-30S, 70S ribosomes with the tetracyclinebound to the 50S subuiiit; 5OS-30S(TC), 70S ribo-somes with the tetracycline bound to the 30S subunit;50S(TC)-30S(TC), 70S ribosomes with tetracyclinebound to both subunits; and 50S-30S, reconstituted70S control ribosomes with no bound tetracycline.

Preparation of poly U, sRNA, and S-100 preincu-bated with tetracycline. The following reaction mix-tures were incubated at 37 C for 10 min: (i) poly U,2.0 mg; tetracycline HCI, 1.0 mg, in 5.0 ml of 0.01 MTris-HCl (pH 7.3) and 0.01 M MgCl2; (ii) sRNA(21), 10.0 mg; tetracycline HCI, 133 ,ug, in 1.0 ml of0.01 M Tris-HCl (pH 7.3) and 0.01 M MgCl2; (iii) S-100protein (19), 23.3 mg; and tetracycline HCl, 233 ,gin a final volume of 1.0 ml. A control for each reac-tion mixture was treated in an identical manner ex-cept that the tetracycline was omitted. Mixtures (i)and (ii) and controls were dialyzed at 3 C againstthree changes of 0.01 M Tris-HCl and 0.01 M MgC12for 8, 16, and 8 hr, respectively. Mixture (iii) and itscontrol were dialyzed twice against 1 liter of 0.02 MTris-HCl (pH 7.9), 0.01 M MgCl2, and 0.005 M mer-captoethylamine for 16 and 4 hr, respectively. Thedialyzed mixtures were removed and frozen in liquidnitrogen until used. The tetracycline-treated compo-nents were designated S-100(TC), sRNA(TC), andpoly U(TC).

Poly U-directed C'4-phenylalanine incorporationstudies. The method of Szer and Ochoa (19) was usedfor experiments involving cell-free polymerization ofphenylalanine with use of poly U as the synthetic

mRNA. The reaction mixtures contained 0.5 mg ofribosomal protein, 1.0 mg of S-100 protein, and 0.75mg of E. coli sRNA in a final volume of 250 ,uliters.The radioactivity of the C'4-phenylalanine incorpo-rated was assayed by the method of Nirenberg (13).

Binding of C'4-phenylalanyl-sRNA by ribosomes.C14-phenylalanyl-sRNA was prepared as describedby Nirenberg, Matthaei, and Jones (15) and had afinal specific activity of 4,650 count/min per OD260.The binding of C'4-phenylalanyl-sRNA to ribosomesin the presence of poly U was achieved by use of themethod of Nirenberg and Leder (14). These reactionmixtures contained, in a final volume of 100 ,uliters,the following: 50 m,umoles of poly U, 2.0 OD2coC'4-phenylalanyl-sRNA, 4.0 OD260 ribosomes, 10Mmoles of Tris-HCl (pH 7.2), 2 ,umoles of Mg acetate,and 5 ,umoles of KCI. The radioactivity was assayedin scintillation vials (14).The sedimentation profile of ribosomal-bound,

C14-phenylalanyl-sRNA was established by use ofdensity gradient centrifugation. The reaction mixturescontained: 1.0 mg of ribosomes, 4.0 OD260 C14-phenylalanyl-sRNA, 5.0 jg of poly U in 200 ,uliters of0.01 M Tris-HCl (pH 7.2) containing 0.01 M MgC12and 0.16 M NH4Cl. The control contained 705 ribo-somes, and the test reaction contained 705(TC)ribosomes. Both mixtures were incubated at 25 C for10 min, and then analyzed by sucrose density gradientcentrifugation as described (3).

Distribution of tetracycline in ribosomal subunitsafter initial binding to either SOS or to 305 particles.The initial reaction mixture contained: 22 mg of 505ribosomes and 16 ,uc of 7-H3-tetracycline (specificactivity of 80 mc/mmole) in 500 uliters of 0.01 M Tris-HCI (pH 7.3), and 0.01 M MgCl2. The mixture wasincubated at 37 C for 30 min. The reaction mixturewas then dialyzed at 3 C for two intervals of 24 hreach against 4 liters of 0.01 M Tris-HCl (pH 7.3) con-taining 0.01 M MgCl2. A 100-Mliter volume of thedialyzed suspension was removed and layered over a4.6-ml sucrose density gradient (5 to 20%) containingTris and Mg+ buffer at the above concentrations.The gradient was centrifuged at 3.5 X 104 rev/minfor 2 hr in the SW-39 rotor. The gradient was frac-tionated and assayed as described (3). The remainderof the dialyzed reaction mixture was then incubatedat 37 C for 30 min with 20 mg of 30S ribosomal sub-units in a final volume of 1.0 ml of 0.01 M Tris-HCl(pH 7.3) buffer containing 0.01 M MgC12. A 100-ulitersample of this reaction mixture was removed andanalyzed by density gradient centrifugation as de-scribed above. The remainder of the reaction mixturewas then dialyzed for two periods of 6 hr each at 3 Cagainst 2 liters of 0.01 M Tris-HCl buffer (pH 7.3)containing 5 X 10-4 M MgCl2 to dissociate the ribo-somal subunits. The entire reaction mixture waslayered over a 54-ml sucrose gradient (5 to 20%) in0.01 M Tris buffer containing 5 X 10-4 M MgC12 andcentrifuged at 2.5 X 104 rev/min for 10 hr in theSW-25.2 rotor of the L2-50 ultracentrifuge. This gradi-ent was fractionated into 1.0-ml fractions, and 50,uliters was removed from each fraction for OD260assay and 50 ,liters was removed for radioactivitydeterminations as described (3). Those fractions con-

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taining the 50S subunits were pooled (Fig. 2C) aswere those containing 30S subunits. These pools werecentrifuged in the SW-39 rotor overnight (19 hr) at3.7 X 104 rev/min. The pellets were taken up in 100,uliters of Tris buffer containing 5 X 1O-4M Mg+,layered over 4.6-ml sucrose gradients (5 to 20%) inthe same buffer, and centrifuged at 3.5 X 104 rev/min.The gradient containing the 50S particles was cen-trifuged for 2 hr; the gradient containing the 30Sparticles, for 4 hr. The gradients were assayed forOD260 and radioactivity (3).The same experiment as described in the previous

paragraph was repeated, except that the initial bind-ing reaction was carried out with 30S ribosomal sub-units and 7-H3-tetracycline. The only variation inprocedure was that the initial series of dialyses of the30S-tetracycline reaction mixture was carried out fora total of 46 hr, the dialyzing buffer being changedafter 6, 16, and 8 hr.

Protein assay. The method of Lowry et al. (12)was used for estimating the protein content of ribo-somal and S-100 suspensions.

Materials. Poly U and tetracycline were the sameas used previously (3). Uniformly labeled C'4-phenyl-alanine with a specific activity of 300 mc/mmoleand 7-H3-tetracycline with a specific activity of 80mc/mmole were purchased from New England Nu-clear Corp., Boston, Mass.

RESULTS

The effects of the tetracycline antibiotics onthe cell-free incorporation of amino acids intopeptides has been documented by others (4,11, 16), and it has been observed in this laboratorythat the amount of tetracycline required to in-hibit the reaction by 50% is about 20 ,ug/mgof ribosomal protein (unpublished data). Thisconcentration varies somewhat depending on theactivity of the extract being used, the more activepreparations being more sensitive to the anti-biotic. To standardize the amount of tetracyclinein the reaction mixtures being assayed, the anti-biotic was preincubated with each componentat the ratio that would normally result in 50%inhibition in the cell-free assay. The resultstabulated in Table 1 indicate that only ribosomeswhich have been pretreated with the antibioticare inhibitory to the cell-free incorporation ofamino acids into peptides, even though it hasbeen observed that the tetracycline binds to thesRNA and to the poly U (3). The preincubationof sRNA both with and without the antibioticresults in a large decrease in the amount of theradioactive label incorporated. We also observedthis when the sRNA and its appropriate controlwere preincubated with tetracycline, and thepolynucleotide and unbound antibiotic wereseparated by gel filtration through SephadexG-25 (unpublished data). The reason for this

TABLE 1. Effect of preincubation of the individualcomponents of the Escherichia coli cell-free

system with tetracycline on the polyU-directed incorporation of C14-phenylalanine into polypeptide

Phenylala-Preincubated component nine Inhibition

incorporateda

miAmoles %S-100 controlb .............. 17.2S-100(TC) ................. 17.3 0

sRNA control ............. 2.8sRNA(TC)........ 3.4 0

Poly U control............ 11.9Poly U(TC) ............... 11.3 5

70S ribosomes............. 12.270S(TC) ribosomes. 6.1 50

a The values tabulated are the average of dupli-cate assays.

bControl components were preincubated andrecovered in the same manner as the tetracycline-treated component except tetracycline was notpresent in the control reaction mixture.

loss of stimulatory activity of the sRNA is notapparent at this time.

Since the data thus far obtained indicated thatthe ribosome was the site of inhibition by tetra-cycline, subsequent studies were directed towardlocalization of this sensitive site on either one orthe other of the ribosomal subunits. Prior data(3) demonstrated that the antibiotic was boundby both the 50S and the 30S subunit; thereforehybrid 70S ribosomes were prepared as describedunder Materials and Methods, and utilized inthe poly U-C'4-phenylalanine assay. These re-sults (Table 2) do not indicate that either sub-unit possesses the sensitive site of tetracyclineinhibition, but rather that tetracycline boundto either unit is equally effective as an inhibitorof this reaction. When the antibiotic is boundto both subunits, the level of inhibition is higherbut is not additive. It is also of interest to notethat the control ribosomes prepared in thismanner are more sensitive to the effects of theantibiotic, since the level that normally resultsin 50% inhibition gives about 80% with theseribosomes.The rapid method of Nirenberg and Leder (14)

for assaying the binding of aminoacyl-sRNA toribosomes in the presence of mRNA was used asa technique to determine if ribosomal-boundtetracycline is as effective as an inhibitor of thisreaction as tetracycline added directly to thereaction mixture. It was also used as an assay

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method, because this reaction does not requirean energy-generating system, as is necessary inthe cell-free peptide synthetic reaction. Table 3indicates that the levels of inhibition with thehybrid ribosomes is about the same as with thecontrol ribosomes to which the inhibitor wasadded at the time the reaction was initiated. Theresults with this assay also suggest that the bind-ing of tetracycline to either one or the other sub-unit is an event which is deleterious to ribosomalfunction, and the sensitivity of these structuresto tetracycline is a characteristic of both. Itshould be pointed out that 2 Mig of tetracyclineadded to the 50S-30S control ribosomes (equiva-

TABLE 2. Effect of tetracycline bound to the 50Ssubunit or to the 30S subunit of Escherichia

coli 70S ribosomes on the poly U-directedincorporation of C'4-phenylalanine

into polypeptide

Phenylala-Ribosomes used nine incor- Inhibition

porateda

mymoles %50S-30S control........... 2.950S-30S control + 10,g

of tetracycline........... .6 7950S(TC)-30S (TC) .......... .9 6950S (TC)-30S ............... 1.2 5950S-30S(TC) ............... 1.4 52

a The values tabulated are the average of dupli-cate assays.

A B C.400

.300-

0.200-

lent to 20 jig of tetracycline per mg of ribosomalprotein) inhibits this reaction by 50%, the levelof inhibition observed when this concentrationof antibiotic is added to the poly U-C'4-phenyl-alanine system. The separation of ribosomalsubunits, preincubation, and recombination toform 70S ribosomes resulted in only a slight lossin activity (about 12%) as evidenced by the com-parison of the amount of radioactivity bound bythe 70S ribosomes (untreated control) and bythe 50S-30S ribosomes.

TABLE 3. Effect of tetracycline bound to the 50Ssubunit or to the 30S subunit of Escherichia coli

ribosomes on the poly U-directed binding ofC14-phenylalanyl-sRNA to

these ribosomes

C'-phenyl-Ribosomes used alanyl-sRNA Inhibition

bounda

count/min S

70S untreated control...... 1,05050S-30S control............ 89650S-305 control + 2,g of

tetracycline .............. 461 49b505(TC)-30S(TC) .......... 413 54505(TC) -30S .............. 384 5750S-305(TC) ............... 429 52

a The values tabulated are the average of dupli-cate assays.bThe per cent inhibition was calculated with the

50S-30S control as 100% incorporation.

D E-4

3

0

I ~~~~~~~~~~~~~0.

10 20 30 40 50 10 20 30

FRACTION No.

FIG. 1. Distribution of radioactivity in reconstituted ribosomes and ribosomal subunits after initial binding of7-H3-tetracycline to 50S subunits. Sedimentation profile of (A) 50S subunits after incubation with 7-H3-tetra-cycline, (B) reconstituted 70S ribosomes after addition of unlabeled 30S subunits to 5OS-7-H3-tetracycline reactionmixture, (C) reconstituted 70S ribosomes after dissociation to 50S and 30S subunits, (D) 50S subunits isolatedfrom dissociated 70S ribosomes [fractions 18 to 23, (C)], and (E) 30S subunits isolated from dissociated 70S ribo-somes [fractions 27 to 35, (C)]. Solid line, OD260; broken line, radioactivity.

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0

C00

CN0

C2

FRACTION No.

FIG. 2. Distribution of radioactivity in reconstituted ribosomes and ribosomal subunits after initial binding of7-H3-tetracycline to 30S subunits. Sedimentation profile of (A) 30S subunits after incubation with 7-H3-tetra-cycline, (B) reconstituted 70S ribosomes after addition ofunlabeled 50S subunits to 30S-7-H3-tetracycline reactionmixture, (C) reconstituted 70S ribosomes after dissociation to 50S and 30S subunits, (D) 50S subunits isolatedfrom dissociated 70S ribosomes [fractions 20 to 24, (C)], and (E) 30S subunits isolatedfrom dissociated 70S ribo-somes [Uractions 28 to 36, (C)]. Solid line, OD260; broken line, radioactivity.

10 20 30 l0 20 30FRACTION No.

FIG. 3. Binding of C14-phenylalanyl-sRNA to Es-cherichia coli ribosomes. (A) 70S control ribosomes,(B) 70S (TC) ribosomes. Solid line, OD260; brokenline, radioactivity.

Since the preparation of ribosomes with theantibiotics bound to either one or the other ofthe ribosomal subunits did not demonstrate a

unique site of inhibition on either one or theother subunit, the suggestion was made that thetetracycline might not remain with the subunitto which it was initially bound during subse-quent manipulations and reconstitution of the70S ribosome. Investigation of this possibilitywas carried out by binding labeled tetracyclineto 50S subunits, adding excess unlabeled 30Ssubunits in 10-2 M Mg++ to reconstitute the70S ribosemes, and then reisolating the individualsubunits to determine the distribution of the

radioactive antibiotic. The results of this study(Fig. 1) indicate that the antibiotic is bound byboth subunits during these manipulations. Theinitial binding reaction was carried out in 10-2 MMg++ because this is the concentration requiredduring the cell-free assay with poly U and phenyl-alanine, although previous studies (3) indicatedmore antibiotic is bound by individual subunitsat 5 X 10-4M Mg++. When the initial bindingwas carried out with the labeled antibiotic andthe 30S particles (Fig. 2), essentially the samefinal distribution of the antibiotic was observedin both subunits. Whether the antibiotic is beingdissociated from the subunit to which it wasinitially bound, making it available for bindingto the other subunit at the time the 70S ribosomesare reformed, or whether excess, unboundantibiotic not removed by extensive dialysis(Fig. IA and 2A) is being bound by the othersubunit, is not clear. The latter seems to be themore likely possibility.The inhibition of binding of aminoacyl-sRNA

to ribosomes by tetracycline antibiotics has beenpreviously reported; Suarez and Nathans (18),using the technique of Nirenberg and Leder(14) and Hierowski (7), established the sedi-mentation profile in a sucrose-density gradient.In both instances, the antibiotic was added di-rectly to the reaction mixture. Figure 3 is theresult of a similar experiment with density-gradient centrifugation; however, the tetracy-cline was preincubated with the ribosomes, theribosomes washed twice to remove unbound

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antibiotic and then used to bind the C14-phenyl-alanyl-sRNA. The 70S(TC) ribosomes bind lessphenylalanyl-sRNA (Fig. 3B) than do the controlribosomes (Fig. 3A).

DIscussIoN

The recent literature (1, 8, 9, 17) indicates thatthe tetracycline antibiotics function by severaldifferent modes of action in the growing cell,depending on the concentration of the antibiotic,i.e., bacteriostatic or bactericidal, conditions ofaeration of the culture, pH of the medium, etc.A series of careful studies by Morrison (1, 8, 9)demonstrated that three modes of action may re-sult in the inhibition of growth of Aerobacteraerogenes. One of these mechanisms was thederangement of protein synthesis; however, it wassuggested that all three modes of action were theresult of inhibition of hydrogen-transfer re-actions. The observation that ribosomal-boundtetracycline is inhibitory to the binding of amino-acyl-sRNA to ribosomes does not lend supportto the view that the interference with proteinsynthesis is the secondary result of inhibition ofhydrogen transfer or other energy-generatingreactions (1, 17). The binding of the aminoacyl-sRNA by ribosomes is not energy-dependent,and the interference with this binding by tetra-cycline suggests that the antibiotic occupies asite on the ribosome which obstructs the attach-ment of the aminoacyl-sRNA, as previouslypointed out by Suarez and Nathans (18).

Since extensive dialysis of the reaction mix-tures described here did not completely removeexcess unbound tetracycline (Fig. 1A and 2A),the possibility exists that the inhibition observedwith the reconstituted ribosomes (Tables 2 and3) might actually result from this unbound anti-biotic acting at some other point. This does notappear to be likely, since the same dialysistechnique was used after preincubation of tetra-cycline with the S-100 fraction, poly U, and thesRNA, and no inhibition was observed in theseinstances, even though unbound tetracyclinewas undoubtedly present after dialysis. Anotherpertinent point is that the 70S(TC) ribosomeswere inhibitory to both the cell-free incorporationof phenylalanine into peptides and to the bindingof C'4-phenylalanyl-sRNA to the ribosomes.These ribosomes were preincubated with theantibiotic and then washed twice through 10%sucrose. It does not seem likely that the excessunbound antibiotic would sediment in this solu-tion and be available as free tetracycline when theribosomal pellet was resuspended in buffer. Onthis basis it is felt that ribosomal binding of theantibiotic is the inhibitory event in the cell-freeprotein synthetic system.

Calculations based on the previous results (3)indicated that about 1 molecule of tetracyclinewas bound to each ribosomal subunit when theseparated subunits were incubated with the anti-biotic in 5 X 10-4 M Mg++. Similar calculationsbased on the data shown in Fig. ID and 1E andFig. 2D and 2E demonstrate the binding of theantibiotic to the individual subunits to be signifi-cantly less than this after extensive dialysis,centrifugations, and other manipulations. Thismay be attributed to the fact that the initialbinding was carried out in 10-2M Mg+-+, andthat these manipulations may have removedsome of the bound antibiotic. About three timesas much tetracycline was bound by the 30S sub-unit as by the 50S, even when the initial bindingreaction was carried out with the 50S particles.It is beyond the scope of this paper to considerpossible mechanisms of inhibition other thanthose concerned with protein synthesis, but it isreasonable to believe that the tetracyclines havemany activities in growing cells in addition tothe interference with protein synthetic reactions.The chelating properties of tetracycline un-doubtedly result in its association with manymacromolecules of the bacterial cell throughmetal complexes (10), and it is therefore difficultto assign to this group of antibiotics a primarylocus of inhibition in the intact cell. The observa-tion that tetracycline binds to both subunits ofE. coli ribosomes (3) and that this ribosomal-bound tetracycline is inhibitory to cell-free pro-tein synthetic reactions and to the binding ofaminoacyl-sRNA to these ribosomes is evidencethat the primary site of inhibition by tetracyclinein the cell-free system is the ribosome, althoughthe location of the inhibitory site has not beenfurther restricted to either the 50S or to the 305subunit by the technique used. Yokota andAkiba (20) reported the isolation of a tetra-cycline-resistant mutant which was also resistantto the antibiotic in cell-free protein syntheticassays. By use of the supernatant enzymes from asensitive strain of E. coli and the ribosomes fromthe resistant culture, it was determined that thesite of resistance was on the ribosome. This typeof mutant can be further utilized to localizethe resistance on the ribosomal subunits, therebyresolving the site of inhibition to one subunitor the other.

ACKNOWLEDGMENTI thank Ernest Grant for his excellent technical

assistance.

LITERATURE CITED1. BENBOUGH, J., AND G. A. MORRISON. 1965. Bac-

teriostatic actions of some tetracyclines. J.Pharm. Pharmacol. 17:409-422.

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2. CONNAMACHER, R. H., AND H. G. MANDEL. 1965.Binding of tetracycline to the 30S ribosomesand to polyuridylic acid. Biochem. Biophys.Res. Commun. 20:98-103.

3. DAY, L. E. 1966. Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracyclineto components of the system. J. Bacteriol. 91:1917-1923.

4. FRANKLIN, T. J. 1963. The inhibition of incorpo-ration of leucine into protein of cell-free sys-tems from rat liver and Escherichia coli bychlortetracycline. Biochem. J. 87:449-453.

5. GALE, E. F., AND J. P. FOLKES. 1963. The assimi-lation of amino acids by bacteria. Biochem. J.53:493-498.

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7. HIEROWSKI, M. 1965. Inhibition of protein syn-thesis by chlortetracycline in the E. coli in vitrosystem. Proc. Natl. Acad. Sci. U.S. 53:594-599.

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12. LOWRY, 0. H., N. J. ROSEBROUGH. A. L. FARR,AND R. J. RANDALL. 1951. Protein measure-ment with the Folin phenol reagent. J. BiolChem. 193:265-275.

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14. NIRENBERG, M. W., AND P. LEDER. 1964. RNAcodewords and protein synthesis. Science 145:1399-1407.

15. NIRENBERG, M. W., J. H. MATTHAEI, AND 0. W.JONES. 1962. An intermediate in the biosynthe-sis of polyphenylalanine directed by synthetictemplate RNA. Proc. Natl. Acad. Sci. U.S.48:104-109.

16. RENDI, R., AND S. OCHOA. 1962. Effect of chlor-amphenicol on protein synthesis in cell-freepreparations of Escherichia coli. J. Biol. Chem.237:3711-3713.

17. SHAPOSHNIKOV, V. N., AND V. K. BAKULEV. 1964.Interrelations between biosynthesis of proteins,of ribonucleic acid, and of certain coenzymes inthe action of tetracyclines on Staphylococcusaureus 209P. Dokl. Akad. Nauk Uz.SSR 158:987-989.

18. SUAREZ, G., AND D. NATHANS. 1965. Inhibitionof aminoacyl-sRNA binding to ribosomes bytetracycline. Biochem. Biophys. Res. Commun18 :743-750.

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