JOURNAL OF BACTERIOLOGY, Oct., 1965Copyright © 1965 American Society for Microbiology

Vol. 90, No 4Printed in U.S.A.

Inhibition of Protein Synthesis byPolypeptide Antibiotics

II. In Vitro Protein SynthesisHERBERT L. ENNIS

Laboratory of Bacteriology, St. Jude Children's Research Hospital, and Department of Biochemistry,University of Tennessee Medical Units, Memphis, Tennessee

Received for publication 1 June 1965

ABSTRACT

ENNIS, HERBERT L. (St. Jude Children's Research Hospital, Memphis, Tenn.).Inhibition of protein synthesis by polypeptide antibiotics. II. In vitro protein syrn-thesis. J. Bacteriol. 90:1109-1119. 1965.-This investigation has shown that the poly-peptide antibiotics of the PA 114, vernamycin, and streptogramin complexes are potentinhibitors of the synthetic polynucleotide-stimulated incorporation of amino acidsinto hot trichloroacetic acid-insoluble peptide. The antibiotics inhibited the transferof amino acid from aminoacyl-soluble ribonucleic acid (s-RNA) to peptide. The Acomponent of the antibiotic complex was active alone in inhibiting in vitro proteinsynthesis, whereas the B fraction was totally inactive. However, the A component,when in combination with the B component, gave a greater degree of inhibition thanthat observed with the A fraction alone. On the other hand, the endogenous incorpora-tion of amino acid was much less susceptible to inhibition than the incorporation of thecorresponding amino acid in a system stimulated by synthetic polynucleotide. In addi-tion, synthesis of polyphenylalanine stimulated by polyuridylic acid was inhibitedto a greater extent when the antibiotics were added before the addition of polyuridvlicacid to the reaction mixture than when the antibiotics were added after the polynucleo-tide had a chance to attach to the ribosomes. However, the antibiotics apparently didnot inhibit the binding of C14-polyuridylic acid or C'4-phenylalanyl-s-RNA to ribo-somes. The antibiotics did not affect the normal release of nascent protein from ribo-somes and did not disturb protein synthesis by causing misreading of the genetic code.The antibiotics bind irreversibly to the ribosome, or destroy the fuinctional identityof the ribosome. The antibiotic action is apparently a result of the competition betweenantibiotic and messenger RNA for a functional site(s) on the ribosome.

A great deal of work has been done to elucidatethe mechanism of protein synthesis in microbialand animal cells. These studies have, in somecases, been aided by the use of antibiotics whichspecifically inhibit a biosynthetic step involvedin protein synthesis. Among these antibiotic in-hibitors of protein synthesis are puromycin(Morris and Schweet, 1961), chloramphenicol(Nathans et al., 1962), streptomycin (Davies,Gilbert, and Gorini, 1964), cycloheximide (Wett-stein, Noll, and Penman, 1964), and the tetra-cyclines (Hierowski, 1965; Suarez and Nathans,1965).

Recently, a new and large group of antibiotics,which apparently inhibit protein synthesis inintact bacterial cells and in cell-free extracts ofthese cells, has been reported. These are thepolypeptide antibiotics of the PA 114, vernamy-cin, and streptogramin group (Celmer and Sobin,1956; Yamaguchi, 1961; Vazquez, 1962; Laskin

and Chan, 1964; Ennis, 1965). As reported in thepreceding investigation (Ennis, 1965), theseantibiotics exist as a complex of synergistic com-pounds. Each antibiotic may be effective itselfin inhibiting growth and protein synthesis, butthey are more effective in combination.The present investigation has shown that

these antibiotics are potent inhibitors of cell-freeprotein synthesis. This report is concerned withthe mechanism of inhibition of protein synthesisby the polypeptide antibiotics. The antibioticsapparently compete with messenger ribonucleicacid (m-RNA) for a functional site(s) on the ribo-some, and in this way stop protein synthesis.

MATERIALS AND METHODS

Bacterial strain. Escherichia coli B was usedas a source of the protein synthesizing system.The bacteria were growni in Brain Heart Infusionbroth (Difco).

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Biochemical and radioactive materials. Di-sodium adenosine triphosphate (ATP), trisodium-guanosine triphosphate (GTP), disodium creatinephosphate, and creatine phosphokinase wereproducts of Calbiochem and of Sigma ChemicalCo., St. Louis, Mo. Polyuridylic acid and thecopolymer of adenylic-cytidylic acid (1:2) werepurchased from Miles Chemical Co., Elkhart,Ind. C14-L-phenylalanine, C14-L-valine, and C"4-L-proline were purchased from New EnglandNuclear Corp., Boston, Mass. E. coli B s-RNAwas a product of General Biochemicals Corp.,Chagrin Falls, Ohio. A Nuclear-Chicago end-window gasflow low-background counter (Nu-clear-Chicago Corp., Des Plaines, Ill.) was usedfor all radioactive counting. Self-absorption wasnegligible.

C14-phenylalanyl-s-RNA was prepared as de-scribed by Von Ehrenstein and Lipmann (1961).Solutions were made freshly from lyophilizedmaterial stored at -20 C.Standard buffer was made up of 0.01 M tris-

(hydroxymethyl)aminomethane (Tris)-HCl (pH7.8), 0.01 M magnesium acetate, 0.06 M KCl, and0.006 M mercaptoethanol (Matthaei and Niren-berg, 1961).

Preparation of cell extracts and ribosomes. Cellextracts (S-30) were prepared as described byMatthaei and Nirenberg (1961). Preincubation,when done, was carried out as described by Davies(1964). The supernatant fraction (S-100) con-taining supernatant enzymes and s-RNA wasprepared from the S-30 fraction by two successivecentrifugations at 100,000 X g for 2 hr in the Spincomodel L-2 centrifuge. Ribosomes were preparedby sedimentation of the S-30 fraction at 100,000X g for 2 hr. The ribosomes were then washedtwo or three times by cycles of sedimentation andresuspension. These ribosomes will not synthesizeprotein unless the supernatant fraction is addedback to the complete reaction mixture. Severalreaction mixtures were used, which are describedin the following sections.

Incorporation of C14-amino acid into hot tri-chloroacetic acid-insoluble peptide. The reactionmixture (1 ml) for the incorporation of C'4-aminoacid into hot trichloroacetic acid-insoluble pep-tide contained: Tris-HCl buffer (pH 7.8), 100,umoles; 2-mercaptoethanol, 6,umoles; magnesiumacetate, 12 MAmoles; disodium creatine phosphate,6 jAmoles; creatine kinase, 20 jug; ATP, 1 jAmole;GTP, 0.12 Mumole; KCl, 100 ,umoles; C'4-aminoacid; S-30 fraction. When measurement of poly-uridylic acid stimulation of the incorporation ofC14-phenylalanine was followed, 50 ,ug ofpolyuridylic acid and 10 m,umoles of C14-phenyl-alanine (0.5 ,uc) were added. Controls lackingpolyuridylate but identical in all other respectsto the reaction mixture described above weresimultaneously carried out. When the copolymercontaining polyadenylic-polycytidylic acid (1:2)was used to stimulate the incorporation of C14-proline, 200,g of polyadenylic-cytidylic acid, 2.5mMumoles of C14-proline (0.5,Mc), and a mixture of

the other 19 nonradioactive amino acids (40 mMA-moles of each) were added, and the S-30 fractionwas preincubated for 45 min at 37 C. The equiva-lent endogenous system lacked only the copolymerof polyadenylic-polycytidylic acid, and the S-30fraction was not preincubated.The reaction was run at 37 C and was terminated

by the addition of 2 volumes of 10% trichloro-acetic acid containing 1 mg/ml of unlabeled aminoacids. The tubes were heated to 95 C for 10 min,cooled, filtered and washed with 5% trichloro-acetic acid on Millipore filters (0.45-,u pore size),mounted on aluminum planchets, and counted. Inthese experiments, unless otherwise stated, thereactions were terminated at 20 min. In the sys-tems stimulated by polyuridylic acid and thecopolymer of polyadenylic-polycytidylic acid,600,u,moles of C14-phenylalanine and 8 MzMAmolesof C14-proline were polymerized. In the corres-ponding endogenous systems, 12 uMMmoles of C'4-phenylalanine and 1.5 s,umoles of C14-proline werepolymerized, respectively. All values were cor-rected for a nonincubated control reaction ter-minated at zero-time.

Synthesis of aminoacyl-s-RNA. The reactionmixture (1 ml) for synthesis of aminoacyl-s-RNA contained: Tris-HCl buffer (pH 7.2), 100Mumoles; magnesium acetate, 13 jumoles; disodiumATP, 1 MAmole; disodium creatine phosphate, 6Mmoles; creatine phosphokinase, 20 Ag; reducedglutathione, 4 MAmoles; E. coli B "stripped" s-RNA, 1 mg; C14-phenylalanine, 2.8 mMumoles (0.5MAc), or valine, 2.5 m,umoles (0.5 Mc); protein of E.coli B S-100, 25 Mg. The reaction was carried outat 30 C. Samples were mixed with an equal voltumeof ice-cold 10% trichloroacetic acid containing 1mg/ml of unlabeled amino acids. The sampleswere filtered and washed on Millipore filters,mounted on aluminum planchets, and countedfor radioactivity.

Transfer of C14-phenylalanine from C14-phenyl-alanyl-s-RNA to hot trichloroacetic acid-insolublepeptide. The reaction mixture (1 ml) for the trans-fer of C14-phenylalanine contained: Tris-HCIbuffer (pH 7.8), 100 Mumoles; 2-mercaptoethanol,6 Mmoles; magnesium acetate, 12 ,moles; disodiumcreatine phosphate, 6 jMmoles; creatine phos-phokinase, 20 jug; disodium ATP, 1 Mumole; tri-sodium GTP, 0.12 Mmole; KCI, 100 Mmoles; S-30fraction (0.575 mg of protein, 0.335 mg of RNA);C'4-phenylalanyl-s-RNA (0.210 mg, 3,120 count/min); nonradioactive phenylalanine, 2 Mumoles;polyuridylic acid, 50,g. The reaction was carriedout at 30 C. Samples were taken and assayed asdescribed for the incorporation of C'4-amino acidinto hot trichloroacetic acid-insoluble peptide.

Binding of C'4-polyuridylic acid to ribosomes.The reaction mixture (1 ml) for the binding ofC14-polyuridylic acid contained: Tris-HCl buffer(pH 7.2), 100 ,umoles; magnesium acetate, 20Mmoles; E. coli ribosomes, washed 2 times (amountas indicated in each experiment); antibiotic ifadded (10 Mg); C'4-polyuridylic acid, 50 Mg, 5,000count/min. The order of addition was as listed.

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The mixture was inculbated at 30 C for 5 min anidthen layered on 4 ml of 0.5 ui sucrose, which hadbeen layered on top of 3 ml of 1.5 M sucrose, in a10-ml Spinco centrifuige tube. The stucrose solu-tions were prepared in the above buffer. Themagnesium acetate was deleted only whenmagnesium was omitted from the reaction mix-tutre. The ttube was centriftuged for 4 hr at 100,000X g, the sucrose was decanted, and the pelletwas carefully washed with stanidard btuffer. Theribosomal pellet was dissolved in concenitratedformic acid, dried oni a planchet, anid counted.

Binding of C14-phenylalanyl-s-RN;VA to ribosomes.The reaction mixtuire (1 ml) for the binding ofC14-phenylalanyl-s-RNA contained: Tris-HCl buf-fer (pH 7.2), 100 ,umoles; maginesium acetate, 20,umoles; KCl, 50 ,umoles; E. coli ribosomes washedtwo times, RNA, 2 mg; antibiotic if added (8,ug/ml); polyuridylic acid, 50 ,g; C14-phenyl-alanyl-s-RNA, 258,ug, 5,840 couint/min. The orderof addition was as listed. The reaction mixturewas incubated at 24 C for 10 min. Less than 10%of the radioactivity bound in the control was hottrichloroacetic acid-insoluble. If antibiotic wasadded, the reaction mixture was warmed at 37 Cfor 2 min after the drug was added, then cooled,and the other components were then added. Thebinding was measured accordinig to the procedureof Nirenberg and Leder (1964).

Antibiotic solutions. The antibiotics used werePA 114 (the crude mixture of A and B), the com-ponents PA 114 A and PA 114 B and vernamycinA and vernamycin Ba, anid streptogramin. Noattempt was made to isolate the components ofstreptogramin. The antibiotics are very insoltublein water and were therefore uised as nonsterilehomogenized suspensions.

RESULTSInhibition of polynridylic acid-stimulated in-

corporation of phenylalanine into hot tricholoro-acetic acid-insoluble peptide. The effect of variousconcentrations of antibiotics on polyuridylicacid-stimulated synthesis of polyphenylalaninein cell-free extracts was determined. Figures 1and 2 show that PA 114, PA 114 A, vernamycinA, and streptogramin were potent inhibitors ofpolyphenylalanine synthesis, inhibiting peptidesynthesis at concentrations as low as 0.1 ,ug/ml.The dose-response curves given in Fig. 1 and2 show that on a weight basis PA 114 A andvernamycin A were better inhibitors than thecrude mixture of PA 114 or streptogramin.Vernamycin Ba (and also PA 114 B), on theother hand, did not inhibit in vitro protein syn-thesis. This finding is in contrast to the results ofthe experiments on intact bacteria described inthe preceding paper (Ennis, 1965).

Polyuridylic acid-stimulated incorporation ofphenylalanine was also inhibited by these anti-biotics in cell-free extracts of rat liver, but the

,00

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STREPTOGRAMIN (0O) OR VERNAMYCIN A (tG.) (pg/ml)

FIG. 1. Inhibition of polyuridylic acid-stimu-lated incorporation of phenylalanine into hot tri-chloroacetic acid-insoluble peptide by streptogramin(0) and vernamycin A (t\). The reaction mixturewas the same as described in Materials and Methodsfor the incorporation of C14-amino acid into hottrichloroacetic acid-insoluble peptide. The S-30fraction contained 0.84 ?ng of RAA and 2.8 mg ofprotein.

100 a E

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10

ANTIBIOTIC (ug/ml)

FIG. 2. Inhibition of polyuridylic acid-stimu-lated incorporation of phenylalanine into hot tri-chloroacetic acid-insoluble peptide by PA 114 (O),PA 114 A (0), and vernamycin B a (A). SeeFig. 1 for other details of experiment.

inhibition was not as great as in the bacteriacell-free extract. For example, 10 ,ug of strepto-gramin per ml resulted in 43% inhibition, and100 Ag only 61%.

Sometimes, for brevity, the results of investi-gations with only one or another of the anti-biotics studied are reported. However, all theexperiments reported in this paper were donewith all the antibiotic mixtures or separate com-ponents, and, unless specifically stated, the re-sults obtained with all the antibiotics were thesame as those obtained with the antibiotic de-scribed.

Synergism of action of mixtures of PA 114 Aand PA 14 B and of vernamycin A and vernamycinBa. A marked synergism between the A and Bfractions of this group of antibiotics on proteinsynthesis in intact cells was demonstrated in theprevious paper (Ennis, 1965). This synergismwas also directed against cell growth. The effect

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of various mixtures of PA 114 A and PA 114 Band of vernamycin A and vernamycin Ba on invitro synthesis of polyphenylalanine was deter-mined. A concentration of the A fraction whichalone would give less than 60% inhibition was

chosen.In one experiment (Table 1), 0.5 ,g/ml of P'A

114 A or 0.5 ,g/ml of vernamycin A was addedto test tubes containing a complete cell-free pro-tein-synthesizing system with polyuridylic acidand C'4-phenylalanine. Amounts of PA 114 B

from 0 to 10 ,g/ml were added to different, tubesof the reaction mixture containing the 0.5,Mg ofPA 114 A or vernamycin A. The 0.5 gg of PA114 A alone resulted in 59 /, inhibition of proteinsynthesis. Addition of 0.1, 0.2, or 0.5 Mug of PA114 B markedly increased the inhibition. Inhibi-tion with 0.5 Mg/ml of PA 114 A and 10 Mg/mlof PA 114 B gave less inhibition than mixtures of0.5 Mug of PA 114 A and 0.5 Mg of PA 114 B3.This was observed in all experiments. Furtherincreases in the amount of the B component ledto even less inhibition, on occasion, than ob-served with P'A 114 A alone. PA 114 B or verna-

mycin Ba alone did not lproduce any inhibition.The experiments with combinations of 0.5

MAg/ml of vernamycin A and PA 114 B (Table 1),and with combinations of vernamycin Ba withPA 114 A or vernamycin A (Table 2), gave simi-lar results.The results of these experiments demonstrated

TABLE 1. Synergistic effect of mixtures of PA 114A and PA 114 B and of vernamycin A and PA

114 B on the inhibition of polyuridylic acid-stimulated incorporation of phenylala-

nine into hot trichloroacetic acid-insoluble peptide*

Per cent inhibition incombination with

PA 114 B addedPA 114 A (0.5 Vernamycin A

pg/ml) (0.5 pg/ml),ug/ ml

0.0 59 230.1 71 370.2 85 550.5 90 7210.0 73 23

* The reaction mixture is that described inMaterials and Methods for the incorporation ofC14-amino acid into hot trichloroacetic acid-in-soluble peptide. The amount of peptide synthe-sized in the preseisce of the various mixtures ofthe antibiotics was compared to the amount ofpeptide made in the absence of antibiotics, to givethe per cent inhibition. The S-30 fraction cois-

tained 0.9 mg of RNA and 1.6 mg of protein.

TABLE 2. Synergistic effect of mixtures of verna-mycin A and vernainycin Ba and of PA 114 Aand vernamycin Ba on the inhibition of poly-

uridylic acid-stimulated incorporation ofphenylalanine into hot trichloroacetic

acid-insoluble protein*

Per cent inhibition incombination with

Vernamycin Ba addedPA 114 A (0.5 Vernamycin A

Vg/mi) (0.5 pg/mi)psg/mi0.0 59 230.1 59 290.2 61 340.5 71 7010.0 74 53

* See footnote to Table 1 for other details ofexperimenit.

TABLE 3. Effect of antibiotics (100 ,Ag/lml) onaminoacyl-s-RNA synthesis*

Aminoacyl-s-RNA synthesized

Antibiotics addedPhenylalanyl-s- Valyl-s-RNARNIA VaysRA

Noise (conitrol). 0.192 0.587PA114A ............ 0.166 0.580Vernamycin A ....... 0.248 0.602Streptogramin ....... 0.248 0.580

* The assay procedture described in Materialsand Methods for synthesis of aminoacyl-s-RNAwas uised. Restults expressed as millimicromolessynthesized per milligram of s-RNA.

that the B coomponent of the antibiotic complexpotentiated the inhibitory effect of the A compo-nent on protein synthesis, even though the Bfraction was inactive by itself. The results fur-ther indicated that PA 114 and vernamycin werevery similar in mode of action. PA 114 B cansynergize with PA 114 A or vernamycin A, andlikewise vernamycin Ba can synergize with eitherPA 114 A or vernamycin A.

This work also showed that the B componentswere not contaminated with the A fraction, be-cause the B component was inactive by itself.

Site of action of the antibiotics. The results givenin Table 3 show that the antibiotics, even at con-centrations as high as 100 ,g/ml, did not inhibitthe synthesis of phenylalanyl-s-RNA or valyl-s-RNA (and thus also activation of these aminoacids), indicating a site of action subsequent tothe formation of aminoacyl-s-RNA.PA 114 A inhibited the rate of transfer of

phenylalanine from phenylalanyl-s-RNA into

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hot trichloroacetic acid-insoluble peptide (Fig.3).

Effect of antibiotics on protein synthesis stimu-lated by synthetic polynucleotides or by endogenousm-RNA. It became evident early during thecourse of this investigation that endogenous pro-tein synthesis was much less susceptible to inhi-bition by the antibiotics than protein synthesis

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FIG. 3. Effect of PA 114 A onsynthesis of hot trichloroacetic acidtide from phenylalanyl-s-RNA. Thture was the same as described inMethods for the transfer of C14-phenC'4-phenylalanyl-s-RNA to hotacid-insoluble peptide. Control, nantibiotic (0); 5 ,ug of PA 114 A p

TABLE 4. Effect of antibiotics (1 ,uridylic acid-stimulated incor)phenylalanine and on endogenozration of phenylalanine into ho,

acetic acid-insoluble pep

Per cent i

Antibiotic added Polyuridylicacid-stimulatedincorporation

C54-phenylalanin

PA 114 .............. 72PA 114 A............ 87Streptogramin ....... 87Vernamycin A 85Puromycin .......... 48

* For reaction mixture used, se(note. The S-30 fraction containedand 1.6 mg of protein.

TABLE 5. Effect of antibiotics (1 ,ug/ml) on the in-corporation of C'4-proline stimulated by thecopolymer of polyadenylic-polycytidylicacid (1:2) and on endogenous incorpo-ration of C14-proline into hot trichloro-

acetic acid-insoluble peptide*

Per cent inhibition

Antibiotic added Polymer-stimulated Endogenous

incorporation incorporation

PA 114 A............ 69 17Streptogramin ....... 82 34Vernamycin A 69 34Puromycin .......... 77 98

* For other details, see footnote to Table 4.

stimulated by synthetic polynucleotides. Table4 shows that 1 ,g/ml of antibiotic inhibited thepolyuridylic acid-stimulated incorporation of Cl4-phenylalanine into hot trichloroacetic acid-in-soluble peptide more than the endogenous incor-poration of the amino acid. Likewise, Table 5shows that the copolymer of polyadenylic-cyti-dylic acid-stimulated incorporation of C'4-prolinewas inhibited to a greater extent than endogenousincorporation of this amino acid. In contrast,puromycin inhibited the endogenous synthesis toa greater extent than the stimulated synthesis.

the kinetics of In these experiments, the synthetic polynu-1-insoluble pep- cleotides were added either after the addition ofe reaction mix- the antibiotic or at exactly the same time. How-Materials and ever, inhibition was decreased if time was allowedtylalanine from for the synthetic polynucleotide to attach to thetrichloroacetic ribosomes and to start functioning before the

to addition of addition of the antibiotic was made. In this ex-)er ml (A). periment, 5 ,tg/ml of PA 114 A were added to a

complete reaction mixture containing polyuri-,g/ml) on poly- dylic acid and C14-phenylalanine either immedi-poration of ately prior to starting the reaction by the addi-us incorpo- tion of the S-30 fraction, or 6.5 min after the re-trichloro- action was proceeding. The curves in Fig. 4 show

tide* that inhibition of peptide synthesis was greaterinhibition of when the antibiotic was added simultaneously

with the polyuridylic acid than after the poly-Endogenous uridylic acid was allowed to attach to the ribo-

incorporation of somes and start functioning.e C154-phenylalanine Effect of antibiotics on the binding of polyuridylic

acid and phenylalanyl-s-RNA to ribosomes. The12 experiments described in the previous section in-30 dicated that perhaps the antibiotics interfered26 with the binding and functioning of m-RNA to36 ribosomes. If the antibiotics were added after the0_ m-RNA-ribosome complex had formed and was

e Table 1 foot- functioning, much less inhibition of protein syn-0.9 mg of RNA thesis was observed than if the antibiotics were

added before the polyuridylic acid attached.

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However, direct measurements of the binding ofC14-polyuridylic acid and C'4-phenylalanyl-s-RNA to ribosomes failed to substantiate thisconclusion. The antibiotics had no effect on theattachment of C14-polyuridylic acid to ribosomes

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TABLE 7. Effect of antibiotics on polyuridylicacid-stimulated binding of C'4-phenylalanyl-s-

RNA to ribosomes*

C14-phenylalanyl-s-RNA bound

Reaction mixture (count/min)

Expt 1 Expt 2

Complete reaction mixture....... 822 608No ribosomes.................... 80No polyuridylic acid............. 92Minus Mg++ t................... 72Plus 0.006 M KC1................ 562Complete (10 min, hot trichloro-

acetic acid) ................... 34Pa 114 A added before polyuri-dylate......................... 750 470

PA 114 added after polyuri-dylate......................... 768

Plus streptogramin.............. - 448Plus vernamycin A . - 416

* Assay procedure was that described in Ma-_______________________ terials and Methods for binding of C14-phenyl-

I0 20 30 alanyl-s-RNA to ribosomes.MINUTES t Actually 0.00012 M Mg+.

FIG. 4. Kinetics of inhibition of polyuridylicacid-stimulated incorporation of phenylalanineinto hot trichloroacetic acid-insoluble peptide byPA 114 A added before or after attachment of poly-uridylic acid. The reaction mixture was the sameas in Fig. 1. The S-SO fraction contained 8.9 mg ofprotein and 1.2 mg of RNA. See text for experi-mental procedure. Control reaction, no antibioticadded (0); 5 ,ug of PA 114 A added at zero-time atthe same time as the polyuridylic acid (l); 5,gof PA 114 A added 6.5 min after starting the re-action (A).

TABLE 6. Effect of antibiotics on C14-polyuridylic acid binding to ribosomes*

C14-polyuridylicacid bound

Reaction mixture (count/min)

Expt 1 Expt 2

Complete reaction mixture....... 777 347Minus Mg++ t................... 68PlusPA 114 A...................... 678 361PA 114 ..368Vernamycin A ........ _ 353Streptogramin ................. 388

* The assay procedure was that described inMaterials and Methods for binding C'4-poly-uridylic acid to ribosomes. In experiment 1, 3mg of RNA were added per ml; experiment 2, 0.5mg of RNA per ml.

t Actually 0.0017 M Mg++.

(Table 6), and little consistent effect of anti-biotics on polyuridylic acid-stimulated binding ofC14-phenylalanyl-s-RNA was observed (Table 7).Although not a consistent finding, occasionally upto 25% inhibition of binding of phenylalanyl-s-RNA was observed (Table 7, experiment 2) when8 ,ug/ml of antibiotic were used, a concentrationgreater than necessary to give maximal inhibitionof peptide synthesis.

Irreversible inhibition of ribosome function. Inthis experiment, the ability of the antibiotics tobind to ribosomes and to destroy their potentialprotein synthetic capability was studied. Com-plete cell-free reaction mixtures without C14-amino acid, as described in Materials andMethods for incorporation of C'4-amino acid intohot trichloroacetic acid-insoluble peptide, wereincubated with or without antibiotics (5 ,ug/ml)for 30 min at 37 C. The ribosomes were thenisolated by centrifugation at 100,000 X g andadded back to complete cell-free reaction mix-tures containing polyuridylic acid, C14-phenylala-nine, and an S-100 fraction, but without anti-biotics. The rate and final amount of C14-phenyl-alanine incorporated into hot trichloroaceticacid-insoluble peptide was determined as outlinedin Materials and Methods for incorporation ofC'4-amino acid into hot trichloroacetic acid-insoluble peptide. In experiment 1, the ribosomeswere washed and then dialyzed overnight against100 times the volume of standard buffer before

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running the reaction. In experiment 2, the ribo-somes were washed once in standard buffer andthen rinsed several times with standard bufferbefore running the reaction. The figures given(Table 8) are per cent inhibition of radioactivityincorporated into hot trichloroacetic acid-in-soluble peptide compared with the control unin-hibited ribosomes, which were run simultaneouslvthrough the same isolation procedure as indicatedin experiments 1 and 2.

Table 8 shows that ribosomes incubated in thepresence of the antibiotics and then washed toget rid of the antibiotics were made incapable ofcarrying out normal protein synthesis in anothercell-free protein-synthesizing system supple-mented with supernatant enzymes and s-RNA.Dialysis of these ribosomes sometimes restoredsome, but not the full, activity of the ribosomes.Portions of the ribosome suspension were addedto complete cell-free protein-synthesizing reac-

tion mixtures to determine whether the impair-ment of ribosome function was due to contamina-tion of the ribosomal suspension by freeantibiotic. The presence of small amounts ofcontaminating antibiotic would be expected toinhibit protein synthesis. No inhibition was ob-served, which indicated that no free antibioticwas present in the ribosome fraction after eitherdialysis or washing.The results indicated that either the antibiotics

were more or less irreversibly bound to a site on

the ribosome, or that the antibiotics destroyedthe functional integrity of the ribosome. Thesealternatives have not been explored.

Competition between PA 114 A and polyuridylic

acid for functional binding to ribosomes. The ex-

periments which have been described so far haveshown that the antibiotics inhibited some func-tional attachment of messenger to the ribosome.If the drugs reached the ribosome before mes-

senger was attached, the subsequent functioningof the ribosome was prevented. This was appar-ently achieved by an irreversible binding to the

TABLE 8. Ability of antibiotics (5 ,ug/ml) to impairsubsequent functioning of ribosomes*

Per cent inhibition ofprotein synthesis

Antibiotic added

Expt Expt 2

PA 114 .................... 74 89

PA 114 A .................. 46 84

Vernamycin A............. 63 85

Streptogramin............. 94 94

* For experimental procedure, see Materialsand Methods.

T.ABLE 9. Inhibition of protein synthesis by PA114, streptomycin, and puromycin at high

concentrations of polyuridylic acid*

Per cent inhibition of protein

Polyuridy lic acid synthesis byadded

PA 114 A Streptomycin Puromycin(0.5 Mg/ml) (7.5 jig/ml) (100 ,ug/ml)

mg/ml0.05 41 87 440.20 36 79 460.50 28 68 521.00 16 61 522.00 11 54 51

* Complete reaction mixtures were supple-mented with polyuridylic acid varying in con-centration from 50,ug/ml to 2 mg/ml. A constantamount of antibiotic was added to each reactiontube. The reactions were run at 37 C for 5 min, andthe C14-phenylalanine incorporated into hot tri-chloroacetic acid-insoluble peptide was de-termined as outlined in Materials and Methods. Acontrol reaction without the antibiotic was runat each concentration of polyuridylic acid.

ribosome, or by destruction of the functionalintegrity of the ribosome. If there is competitionbetween m-RNA and antibiotic, one would ex-pect that the extent of inhibition of proteinsynthesis would be dependent on the amount ofm-RNA competing with the antibiotic for thefunctional site(s) on the ribosome. The moremessenger present in the system, the greater thechance would be that the messenger moleculewould attach to the functional site(s) on theribosome before the antibiotic got there.

Reaction mixtures containing 0.5 ,ug/ml of PA114 A and increasing amounts of polyuridylicacid were analyzed for their ability to incorporatephenylalanine into hot trichloroacetic acid-in-soluble peptide. Control reactions lacking theantibiotic but containing the polyuridylic acid ateach concentration were run at the same time.The inhibition was calculated by dividing theamount of phenylalanine incorporated in the re-actions containing antibiotic by the amount in-corporated by the corresponding reactions con-taining the same amount of polyuridylic acidbut no antibiotic. Table 9 shows that the morepolyuridylic acid present, the less inhibition wasobserved, indicating that the polynucleotide wasable to compete with the antibiotic.

Table 9 also shows that the same result was ob-tained with streptomycin, which is also known tobind irreversibly to the ribosome (Flaks, Cox,and White, 1962), but which has a different modeof action than PA 114 A. No decrease in inhibi-tion was observed when puromycin was used to

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inhibit polyphenylalanine synthesis. Puromycindoes not bind irreversibly to the ribosome (Mor-ris et al., 1963).

Further observations. The effect of the anti-biotics on the release of nascent protein chainsfrom ribosomes was studied. A complete cell-freereaction mixture containing polyuridylic acidand C'4-phenylalanine, as described in Materialsand Methods for incorporation of C'4-amino acidinto hot trichloroacetic acid-insoluble peptide,was incubated for 5 min at 37 C. The reactionmixture was cooled rapidly and centrifuged at100,000 X g for 2 hr to sediment the ribosomes.The ribosomes were rinsed with cold standardbuffer and resuspended in standard buffer. Theseribosomes (with attached nascent chains of C14_polyphenylalanine) were then incubated in a com-plete reaction mixture containing nonradioactivephenylalanine (4 ,umoles/ml) and an S-100 frac-tion. Antibiotics were added to separate tubes ofthis mixture as indicated, and samples weretaken at zero-time and after 5 min of incubationat 37 C. The samples were chilled rapidly, andthe ribosomes were reisolated and washed asabove, dissolved in concentrated formic acid,dried on planchets, and counted for radioactivity.The reaction mixture contained (per milliliter)0.125 mg of ribosomal RNA and 0.3 mg of super-natant protein.The results given in Table 10 show that the

antibiotics had very little effect on the release ofnascent protein from ribosomes. The sameamount of protein was released after 5 min in thepresence or in the absence of the antibiotics. Ashas been already shown (Morris and Schweet,1961), puromycin caused a premature release ofnascent protein from ribosomes in this system.

Davies et al. (1964) showed that streptomycindisturbs protein synthesis by causing a misread-ing of the genetic code. In one series of experi-ments, 50 mg/ml of polyuridylic acid were added

TABLE 10. Effect of antibiotics (10 /g/ml) on releaseof nascent protein from ribosomes*

Antibiotic added C14-phenylalanine associatedwith ribosomes

count/minNone (zero-time). 210None (5-min incuba-

tion) ................ 185PA 114................ 171PA 114 A.............. 162Vernamycin A 150Streptogramin ......... 155Puromycin (100 gg/ml) 68

* See text for experimental procedure.

to a complete cell-free protein-synthesizing sys-tem containing a complete mixture of nonradio-active amino acids and, depending on the experi-ment, a variety of radioactive amino acids. Inanother experiment, the endogenous protein-synthesizing system was used, and a radioactivealgal amino acid mixture was added. The experi-ments were run at Mg++ concentrations varyingfrom 2 to 30 ,moles/ml. In no case did any ofthe antibiotics induce a specific misreading of thegenetic code.

DISCUSSIONA previous investigation (Ennis, 1965) showed

that the polypeptide antibiotics of the PA 114,vernamycin, and streptogramin complexes spe-cifically inhibit protein synthesis in intact bac-terial cells. The present work extends this resultto the in vitro synthesis of protein. However,these antibiotics are potent inhibitors only of thesynthetic polynucleotide-stimulated incorpora-tion of amino acids into hot trichloroacetic acid-insoluble peptide. The endogenous incorporationof amino acid is much less susceptible to inhibi-tion than the corresponding stimulated system.The antibiotics inhibit protein synthesis well

only when m-RNA is not attached to the ribo-somes and is functioning. If the antibiotics areadded after the messenger is firmly attached andfunctioning, inhibition is less than when the anti-biotics are added before m-RNA becomes at-tached and is working. This indicated that theantibiotics might prevent the binding and func-tioning of m-RNA. However, direct measure-ments of C14-polyuridylic acid binding to ribo-somes in the absence of protein synthesis showedthat physical attachment of polyuridylic acid toribosomes is unaffected by concentrations of theantibiotics 10-fold greater than that required tomaximally inhibit in vitro protein synthesis.However, another problem still exists. Althoughthe antibiotics do not affect attachment ofm-RNA (and also of aminoacyl-s-RNA), they doinhibit the functioning of the m-RNA-amino-acyl-s-RNA-ribosome complex formed after, butnot before, the antibiotic is added to the in vitroprotein synthesizing system. The drugs thereforedo not compete with the site(s) necessary to thephysical binding of m-RNA or aminoacyl-s-RNA.They may compete, however, with some othersite(s) necessary for the functioning of the ribo-some-m-RNA complex. Thus, there may be onesite on the ribosome responsible for the physicalattachment of messenger and others for func-tional attachment.The results presented in this investigation

show that the antibiotics do indeed compete withm-RNA for a functional site(s) on the ribosome.

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The first piece of evidence bearing on this pointindicates that the antibiotics bind irreversibly tothe ribosome, or destroy the functional integrityof the ribosome when the ribosome is free frommessenger. Ribosomes were incubated in a com-plete reaction mixture with unlabeled aminoacids and antibiotic, then isolated by centrifuga-tion, resuspended, and tested for the ability tosupport protein synthesis in the absence of anti-biotic. Little protein synthesis was observed,indicating that the ribosomes were nonfunctional,owing to the preincubation with the antibiotic.The second line of evidence gave direct evidencethat the antibiotics compete with m-RNA. Re-action mixtures containing a constant amount ofantibiotic and increasing amounts of polyuridylicacid were analyzed for their ability to synthesizepolyphenylalanine. The reaction mixtures con-taining a high level of polyuridylic acid were in-hibited to a lesser extent than reactions contain-ing small amounts of the polynucleotide.

These findings lead to the conclusion that theantibiotic action is due somehow to the competi-tion between antibiotic and m-RNA for a func-tional site(s) on the ribosome. The exact mecha-nism by which this occurs is, however, not known.

Streptomycin also competes with polyuridylicacid for a site(s) on the ribosome, but its mode ofaction is different from that of the polypeptideantibiotic studied. These polypeptide antibioticsand streptomycin may belong to the same class ofantibiotic inhibitors of protein synthesis, in thatthey both can bind irreversibly to a functionalsite(s) on the ribosome. The binding may leadto a change in the structure of the ribosome,resulting in an impaired ability to carry outprotein synthesis. Streptomycin and the poly-peptide antibiotics may even bind at the samesite(s), but the degree to which each antibioticaffects the functioning of the ribosome may bedifferent. In the case of streptomycin, the changein the structure of the ribosome causes a mis-reading of the genetic code (Davies et al., 1964).The effect of the polypeptide antibiotics is moresevere, resulting in a complete cessation of pro-tein synthesis.The antibiotics of the PA 114, vernamycin,

and streptogramin complexes inhibit growth andprotein synthesis in intact cells of Bacillus subtilisand Staphylococcus aureus, but are relatively in-active against intact E. coli cells, inhibitinggrowth only slightly at very high concentrations(English, McBride, and Halsema, 1956; Tanakaet al., 1958; Verwey, West, and Miller, 1958).The fact that the antibiotics inhibit in vitro pro-tein synthesis indicates that E. coli cells areprobably impermeable to the drugs. When theimpermeability barrier is removed by making an

extract, the antibiotics can act and inhibit pro-tein synthesis. However, when Yamaguchi andTanaka (1964) were working only on the inhibi-tion of endogenous synthesis of protein in cell-free extracts of E. coli by mikamycin, an anti-biotic which is probably the same as or similarto the PA 114 group (Yamaguchi, 1961; Wa-tanabe, 1961), they found, as the present studyindicates, that the E. coli endogenous system it-self was relatively insensitive to inhibition by thisantibiotic. From this result, Yamaguchi andTanaka (1964) concluded that the mechanismof protein synthesis may be different in gram-positive and gram-negative bacteria. This con-clusion must be reviewed in view of the presentfinding that in vitro endogenous protein synthesisin E. coli extracts is not as susceptible to inhibi-tion by the polypeptide antibiotics as is a systemstimulated by synthetic polynucleotides. Thepresent study has shown that the in vitro pro-tein-synthesizing system derived from E. coli isrefractory to inhibition only when m-RNA isattached to the ribosomes before the antibioticis added to the reaction mixture. The results ob-tained by Yamaguchi and Tanaka (1964), con-trary to their conclusion that the E. coli protein-synthesizing system is not susceptible to inhibi-tion by mikamycin, gives further evidence thatthe relative insensitivity of intact E. coli cells toinhibition by these polypeptide antibiotics of thisgroup is due to the impermeability of the cell tothese antibiotics.The antibiotics of the PA 114 and vernamycin

group are composed of a number of components.Each component may be active alone in inhibitingbacterial growth and protein synthesis in intactgram-positive bacterial cells, but they are muchmore active in combination with each other(Celmer and Sobin, 1956; English et. al., 1956;Tanaka et. al., 1958; Yamaguchi, 1961; Ennis,1965). Polyuridylic acid stimulation of the syn-thesis of polyphenylalanine in extracts of E. coliis very sensitive to inhibition by the crude mix-tures of PA 114 or streptogramin, and by PA 114A or vernamycin A. In contrast to the resultsobtained with intact cells, PA 114 B or vernamy-cin Ba, even at high concentration, does notinhibit in vitro protein synthesis. Laskin andChan (1964) also noted the inactivity of verna-mycin Ba. However, a marked synergism be-tween the A and B fractions of these antibioticson in vitro protein synthesis was demonstrated.For example, in one experiment, 0.5 ,g/ml of PA114 A gave 59% inhibition of polyphenylalaninesynthesis. Addition of 0.5 ,ug/ml of PA 114 B(which alone gives no observable inhibition) re-sulted in an increase in inhibition to 90%. Thisindicates that the B component, although inac-

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tive by itself, can potentiate the effect of the Acomponent.

Heterologous mixtures of PA 114 A and verna-mycin Ba and vernamycin A and PA 114 B giveresults similar to those produced by homologousmixtures of PA 114 A and PA 114 B or verna-mycin A and vernamycin Ba. Together withother biological and chemical data, these re-sults give further support to the idea that theseantibiotics are either the same or very similar.

If the concentration of PA 114 A (or vernamy-cin A) is kept constant at 0.5 jig/ml and theamount of PA 114 B (or vernamycin Ba) isvaried, a point is reached beyond which no fur-ther increase in inhibition of protein synthesis isobserved. After this point, further addition of PA114 B results in even less inhibition than thatobtained with lower concentrations of PA 114 B.Occasionally this inhibition is less than that ob-served with PA 114 A alone. The reason why thisoccurs has not been studied. Perhaps, at highconcentrations, PA 114 B competes with PA 114A for sites on the ribosome and replaces PA 114A on the ribosome. Since PA 114 B alone is in-active in inhibiting protein synthesis, a diminu-tion in inhibition results. Laskin and Chan (1964)stated that vernamycin A does not synergizewith vernamycin Ba in inhibiting cell-free syn-thesis of polyphenylalanine. The results of thepresent investigation contradict these findings.Perhaps these investigators used too high a con-centration of vernamycin A to start with. If in-hibition is much greater than 60% to begin with,little enhancement of this inhibition is observedwhen vernamycin Ba is added.The polypeptide antibiotics of the PA 114,

vernamycin, and streptogramin complexes arefurther examples of antibiotics which inhibitgrowth of bacterial cells by specifically inhibitingprotein synthesis. They are a new class of com-pounds, because their mode of action, althoughnot yet fully understood, appears to be differentfrom the antibiotics which have been studiedby others. Puromycin inhibits protein synthesisby causing premature release of nascent peptidefrom ribosomes (Morris and Schweet, 1961);streptomycin, by causing a misreading of thegenetic code (Davies et al., 1964); the tetracy-clines, by inhibiting binding of aminoacyl-s-RNAto ribosomes (Hierowski, 1965; Suarez andNathans, 1965); and chloramphenicol (Nathanset al., 1962), by a poorly understood mechanismwhich is, however, different from that of the PA114 group. The cross-resistance of PA 114 resist-ant mutants with streptogramin and vernamy-cin, and with erythromycin and oleandomycin(Ennis, 1965), perhaps indicates a similar modeof action.

This work has shown that the mode of actionof the PA 114, vernamycin, and streptogramincomplexes is the same. Other investigations onthe biological effects of the antibiotics and ontheir chemical structure have also indicated amarked similarity among this group and amongother polypeptide antibiotics such as mikamycin,ostreogrycin, and staphylomycin (Celmer andSobin, 1956; English et al., 1956; Verwey et al.,1958; Tanaka et al., 1958; Eastwood, Snell, andTodd, 1960; Vanderhaeghe and Parmentier,1960; Yamaguchi, 1961; Watanabe, 1961; Vaz-quez, 1962; Bodansky and Ondetti, 1963; Laskinand Chan, 1964).

ACKNOWLEDGMENTS

I wish to thank W. D. Celmer, Chas. Pfizer &Co., Inc., for the PA 114, PA 114 A, and PA 114B; R. Donovick, Squibb Institute for MedicalResearch, for the vernamycin A and vernamycinBa; F. J. Wolf, Merck Sharp & Dohme, for thestreptogramin; and Mrs. R. Tirey for her assist-ance in some of the experiments.

This investigation was supported by PublicHealth Service grant GM 12359-01 from the Divi-sion of General Medical Sciences, by grant GB-2399 from the National Science Foundation, andby the American Lebanese Syrian AssociatedCharities (ALSAC).

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