Insights into Protein Biosynthesis and Ribosome Function through Inhibitors

.

I

SIDNEYPESTKA Roche lnstitute of Molecular Biology Nutley, New Jersey

-

I. Introduction * . . . * * 11. Summary of Protein Biosynthesis . . . . . . . . 111. Translocation Inhibitors: Localization of Action through Nonenzymic and Enzymic Translocation IV. Ribosomal States . . . . . . . . . . . V. Erythromycin Binding to Ribosomes . . . . . . . VI. Effect of Chloramphenicol on the Puromycin Reaction: Models of Ribosome Function . . . . . . . . . . . VII. The Sistrand: The Translational Unit . . . . . . . References. . . . . . . . . . . . .

. . . . . . . .

217 217 223 223 230 234 238 244

1. Introduction This account is a highly personal view of some selected experiences with antibiotic inhibitors of protein synthesis. It is not intended to be a comprehensive review of the literature or of my own work. It is intended to outline some interesting, perhaps even controversial, observations and interpretations that have emanated from studies of inhibitors of protein biosynthesis. In general, detailed knowledge of protein biosynthesis has enabled localization of the action of many antibiotics and other inhibitors. Nevertheless, examination of the effects of various inhibitors on steps of protein biosynthesis has uncovered intriguing and valuable insights into protein synthesis as well as into ribosome structure and function.

II. Summary of Protein Biosynthesis For reference, the steps of protein biosynthesis are summarized in Fig. 1. The details of these interactions are summarized in the legend to Fig. 1. For further details, a number of recent reviews may be consulted (I, 2). Inhibitors of protein synthesis can be classified in a number of ways (35). According to the classification presented in Table I, inhibitors are divided into groups according to their site of inhibition: supernatant factors, small subunit (30 S or 40 S ) or large subunit (50 S or 60 217

INITIATION

RECOGNITION OF INTERNAL CODONS

/.-" .

.

*-=

E

PEPTIDE BOND FORMATION. TRANSLOCATION

P

-

TERMIN"

am a o

n

P

INHIBITORS OF PROTEIN SYNTHESIS

219

S ) . Then they are classified according to their ability to inhibit these functional sites in prokaryotes only, in eukaryotes only, or in both proand eukaryotes. The classification is simplified in that inhibition of mitochondrial or chloroplast ribosomes is not included as a distinct entity. In general, although most of the prokaryotic inhibitors block protein synthesis in bacteria, blue-green algae, mitochondria, and chloroplasts, some show some specificity within this group. For example, erythromycin and lincomycin inhibit protein synthesis by bacteria, but not by rat liver mitochondria ( 6 ) . Likewise, some inhibitors exhibit some discrimination among various eukaryotes ( 7 ) . The puromycin reaction (summarized in Fig. 2 ) has been extremely useful in studying transpeptidation and even other steps of protein biosynthesis. In the presence of puromycin, which serves as an analog of aminoacyl-tRNA, donor peptides or peptide analogs on peptidyl-tRNA can be transferred to puromycin in the presence of ribosomes. The peptidyltransferase that catalyzes this reaction is an integral part of the ribosome structure. Donors for the puromycin reaction may be peptidyltRNAs of many sorts. For example, native peptidyl-tRNA may be used on polyribosomes isolated from intact cells. Synthetic peptidyl-tRNAs, such as polyphenylalanyl-tRNA, polylysyl-tRNA, N-acetylphenylalanyltRNA, or formylmethionyl-tRNA, may serve as suitable peptidyl donors FIG.1. Schematic summary of protein synthesis. The semilunar cap ( I ) represents the free 30 S subunit. Initiation of protein synthesis involves attachment of mRNA to a 30 S subunit ( I ) to form complex 11; this process requires Mg” as well as initiation factor IF-3. Subsequent attachment of fMet-tRNA, in response to initiation codon AUG, to form complex I11 requires GTP and initiation factors IF-I and IF-2. Junction of the 50 S subunit to complex 111 produces complex IV. Enzymic recognition of internal codons involves factors EF-Tu, EF-Ts, and GTP: the (EFTu) GTP * ( Ala-tRNA) complex binds to the ribosomes in response to the GCU codon to form complex V. Peptide bond formation occurs by transfer of the fMet (peptidyl) group to form fMet-Ala ( V I ) ; peptidyl transfer requires only ribosomes and K . Translocation involves several coordinate processes, such as: release of deacylated tRNArMetto form state VII; one-codon movement of mRNA and ribosome with respect to each other, precisely positioning the next codon, UCU, into position for translation; and coordinate movement of peptidyl-tRNA ( fMet-Ala ) from the “A” to “P” site, resulting in state VIII. By repetition of the codon recognition step, Ser-tRNA would enter the A site in response to the codon UCU. Complex IX represents a peptidyl-tRNA with a polypeptide almost completed. Transpeptidation and translocation produces complex X with a completed protein still attached to tRNA and a termination codon, UAA, in the next recognition site. In response to a release factor, the completed protein is released, and perhaps also tRNA after translocation (XI). Provided no further sistrands (see Fig. 22) are to be translated, the ribosome may be dissociated into 30 S and 50 S subunits with release of mRNA (XII). Alternatively, mRNA may be degraded prior to this stage. See reviews (1, 2 ) for further details.

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TABLE I CLASSIFICATION OF INHIBITORS OF PROTEIN SYNTHESIS Supernatant Folk acid antagonists

Aminoalkyl adenylates Guanosine B’-(B,y-methylene)triphosphate Fusidic acid

Diphtheria toxin

30 S (40 S)

Prokaryotic inhibitors Aminoglycosides Streptomycin Dihydrostreptomycin Paromom ycin Neom ycin Kanam ycin Gentamycin Bluensomycin y m y c i n asugamycin Negam ycin Edeine Edeine A Edeine B

50 S (60 S)

Chloramphenicol Macrolides Niddamycin Carbomycin Spiramycin I11 Tylosin Leucomycin Erythromycin Chalcomycin Oleandomycin Lankam ycin Meth ym ycin Lincomycin Streptogramin A group Ostreogrycin A PA114A Streptogramin A Vernamycin A Mikamycin A Streptogramin B group Ostreogrycin B PA114B, etc. Viridogrisein (etamycin) Thiostrepton group Thiostrepton (bryamycin, thiactin) Siomycin A Thiopeptin B Multhiomycin Sporangiomycin A-59 Althiomycin Micrococcin Bottromycin A2

Both Prokaryotic and Eukaryotic Pactamycin Aurintricarboxylic acid

Eukaryotic Inhibitors Harringtonine alkaloids

Purom ycin 4-Aminohexose pyrimidine nucleosides Gougerotin Amicetin Blaaticidin S Plicacetin Bamicetin Sparsomycin Tetracyclines Chlortetracycline Glutarimides Cycloheximide (Actidione) Acetoxyc ycloheximide Streptovitacin A Ipecac alkaloids Emetine Anisomycin Trichodermin

221

INHIBITORS OF PROTEIN SYNTHESIS PEPTIDE

f!3

PUROMYCIN

PEPTIDYLTRANSFERASE

PEPTIDYLPUROMYCIN

FIG.2. Schematic illustration of the puromycin reaction with peptidyl-tRNA on ribosomes or polyribosomes.

( 3 ) . Furthermore, the oligonucleotides that comprise the ends of these aminoacyl-tRNAs also may be suitable donors. For example, C-AC-C-A ( AcPhe ) or C-A-A-C-C-A ( f Met ) , the aminoacyl-oligonucleotide termini of AcPhe-tRNA and fMet-tRNA, respectively, can serve as suitable donors (8). Similarly, acceptors of the puromycin reaction may be any aminoacyl-tRNA or a number of aminoacyl-tRNA analogs as shown in Fig. 3. Puromycin and the puromycin analogs are the smallest acceptors described. Analogs of aminoacyl-tRNA, such as C-A-C-C-A(Phe ) and similar compounds, also serve as suitable acceptors. Many puromycin analogs that shed light on the structural requirements of the acceptor have been made. For example, hydroxypuromycin (Fig. 3 ) can serve as an acceptor, which indicates that transfer can be made to a hydroxyl as well as an amino group (9, 10).In addition, two unique carbocyclic analogs of puromycin have been synthesized (Fig. 3 ) and their activity in various assays has been determined (10). Studies with these analogs have indicated that neither the sugar moiety CHs,

NH

I

OH

c=o I

PUROMYCIN

NH

I

OH

c=o I

JI-HYDROXYP U R O M YC I N

NH

I

c=o I

OH

HO

C , H3 N

NH

I

c=o I

CARBOCYCLIC P U R O M Y C I N A N A L O G S

FIG.3. Structures of puromycin, +hydroxypuromycin ( methoxy group is absent), and carbocyclic puromydn analogs I and I1 ( stereoisomers, with hydroxymethyl group absent).

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nor the 5-hydroxymethyl group is necessary for puromycin to serve as a suitable acceptor. Furthermore, carbocyclic-puromycin analog I was 100-fold more active than analog 11. Further studies on these carbocyclicpuromycin analogs should be enlightening. An example of the effect of carbocyclic analog I on peptidyl puromycin synthesis with polyribosomes is shown in Fig. 4. It can be seen that the carbocyclic puromycin analog inhibits peptidyl-puromycin synthesis with a K , of 10 pM. As expected, the inhibition is completely competitive with puromycin, which under these same conditions has a K, of 4 pM. The similar reaction with hydroxypuromycin provides a K i of 200 pM for hydroxypuromycin. It is also noteworthy that puromycin has about 100-fold greater affinity for polyribosomes than for 1 M NH,C1-washed ribosomes as judged by the K, for the reactions (11). 10 9

8

7

I

4

6

5 4 3

2 I

0

2

4

6

0

1

0

[PUROMYCIN]-'X~O-~

[CARBOCYCLIC PUROMYCIN]xlOS

FIG.4. Competitive inhibition of peptidylpuromycin synthesis by Esckrichio cob polyribosomes with carbocyclic analog I of puromycin. Each 50-al reaction mixture contained 50 mM Tris-Ac ( p H 7.2), 100 mM KCI, 4 mM MgCl, and 2 Amounits of E. c d i polyribosomes ( 1 0 ) . [aH]Puromycin and the carbocyclic analog of puromycin were present at the concentrations indicated in the figure. Reaction mixtures were incubated at 24°C for 1 minute and assayed as described (10). In the left panel, a double-reciprocal plot of the data is presented. For each curve of the left panel, the concentration of the carbocyclic analog of puromycin used is given. The KI of 8-11 pM was calculated with the use of a K, of 4.45 pM for puromycin under these conditions represented by the data in the absence of the carbocyclic analog. In the right panel, a Dixon plot of the data is presented. The Ki in this case was determined to be 11 pM. The various amounts of ['H]puromycin used in the reactions are shown in the figure. The interaction coefficients obtained for carbocyclic analog I of puromycin range from 0.8 to 1.0; these were obtained from Hill plots of the data.

INHIBITORS OF PROTEIN SYNTHESIS

223

111. Translocation Inhibitors: Localization of Action

through Nonenzymic and Enzymic Translocation Although the mechanism of nonenzymic translocation has not yet been delineated (12,13),studies on the effect of antibiotics on enzymic and nonenzymic translocation have proved to be simple and powerful techniques for localizing antibiotic action ( 12). That nonenzymic translocation is, in fact, distinct from enzymic translocation has been demonstrated (12,13). The essential difference between enzymic and nonenzymic translocation is the need for elongation factor EF-G for enzymic translocation, but not for the nonenzymic reaction. This was demonstrated in several ways. When proteins L7 and L12 were removed from ribosomes, enzymic translocation was inhibited, but nonenzymic translocation was not affected. Also, thiostrepton inhibited nonenzymic translocation from ribosomes devoid of L7 and L12 (12).Furthermore, p-chloromercuribenzenesulfonate, which inhibits elongation factor EF-G and thus is a powerful inhibitor of enzymic translocation, had no effect on or stimulated nonenzymic translocation (12,13). I n addition, guanosine 5'- ( p,y-methylene ) triphosphate, a competitive inhibitor of GTP, inhibited enzymic, but not nonenzymic translocation. Furthermore, when ribosomes and EF-G were prepared from an E . coli strain containing a temperature-sensitive EF-G, enzymic translocation was temperature sensitive, but nonenzymic translocation was not ( 12). Nevertheless, although the mechanism of nonenzymic translocation is not yet understood, it can be and has been used successfully to localize antibiotic action. For example, any agent that inhibits both enzymic and nonenzymic translocation must be a ribosomal inhibitor, for nonenzymic translocation requires only ribosomes, but no additional supernatant factors. In this way, thiostrepton, siomycin A and micrococcin were determined to be ribosomal inhibitors (12, 14, 15). Any agent that inhibits the enzymic, but not the nonenzymic process, must therefore be an inhibitor of elongation factor EF-G. In this way, fusidic acid was demonstrated to be an inhibitor of EF-G (16).These results are summarized in Table 11. Thus, by simple and direct comparison of the effect of an antibiotic on enzymic and nonenzymic translocation, one can localize the action of an antibiotic to either EF-G or to the ribosome. Mixing experiments with EF-G and ribosomes from antibiotic-sensitive and -resistant strains are therefore unnecessary. For more detailed studies of these inhibitors, recent reviews can be consulted (17,18).

IV. Ribosomal States During protein synthesis, ribosomes exist in a number of discrete functional states, each state containing the attached components differ-

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TABLE I1

CLASSES OF INHIBITORS OF TRANSLOCATION‘

Antibiotic Fusidic acid Thiostrepton, siomycin A Micrococcin

Cell component

RiboNonsome: Enzymic enzymic mRNA GTP TransAAtranshypepti- tRNA trans- movelocation location ment drolysis dation binding

EF-G

+b

-

Ribosome Ribosome

+ +

+ +

+ + +

+ + k

-

-

-

+ +

a Although these substances inhibit various translocation reactions, thiostrepton, siomycin A and, perhaps, micrococcin can also inhibit aminoacyl-tRNA (AA-tRNA) binding, which may, in fact,, be the predominant effect on intact cells. Also, micrococcin may have some effect on GTP binding to ribosomes. b Inhibition; -, no effect; f,pmsible effect.

+,

Ia

EFO,

FIG.5. Ribosomal states and the ribosome cycle. The individual complexes I-VIII are described in the legend to Fig. 1. The initiation sequence comprises stages I-IV. For every addition of a single amino acid (i.e., elongation), the ribosome cycles from state V to VIII. State IV is formally equivalent to VIII. Thus, the elongation epicycle (V-VIII) is superimposed on the overall ribosome cycle and repeats itself on each amino-acid addition. The termination sequence is not given in detail, as it has not been precisely delineated. Dissociation factor ( D F ) bound to 30 S subunits ( l a ) may be identical to one of the initiation factors and may be involved in dissociation of 70 S ribosomes after termination. The symbol “f” represents fMet.

INHIBITORS OF PROTEIN SYNTHESIS

225

ently juxtaposed. It is very likely that each functional state corresponds to a conformational state as well. Ribosomes can exist as free subunits, as a 30 S subunit initiation complex and as various 70 S states depending on the location and presence of aminoacyl-tRNA, peptidyl-tRNA or deacylated-tRNA (states IV-VII) as illustrated in Fig. 5. During each elongation epicycle-that is, during the addition of a single amino acidthe ribosomes pass through states IV-VII. Each elongation epicycle takes place on the polyribosome. Although it is clear that the various ribosomal states exist, it was not clear until recently that antibiotics discriminate between various states. Inhibitors that discriminate between the various states have been designated topostatic agents (19). Topostatic agents are agents that inhibit or stimulate a reaction depending on the state of the components involved. For example, a number of antibiotics can inhibit peptide bond formation when AcPhe-tRNA is a donor. AS illustrated in Table I11 under the column “Synthetic Models for Peptide Bond Synthesis” and in Table IV, chloramphenicol, sparsomycin, the aminonucleosides ( amicetin, gougerotin and blasticidin S ), lincomycin, and macrolides (erythromycin, niddamycin, carbomycin, tylosin and spiramycin 111) as well as streptogramin A antibiotics (PA114A and vernamycin A ) were excellent inhibitors of synthetic models for peptide synthesis (3, 15, 20). In these assays, washed ribosomes and the synthetic donor AcPhe-tRNA was used. Althiomycin did not inhibit this reaction under the conditions studied ( 1 5 ) , but could inhibit acetylphenylalanylpuromycin synthesis when conditions were varied (21, 22). In striking contrast were the results when native polyribosomes were used for the assay of peptidylpuromycin synthesis (Tables I11 and IV). Chloramphenicol, sparsomycin, and the aminonucleosides were good inhibitors of peptidylpuromycin synthesis (23). They were good inhibitors of both the synthetic as well as the native reaction. However, lincomycin and the macrolides as well as the streptogramin A antibiotics were ineffective in inhibiting peptidylpuromycin synthesis. Furthermore, althiomycin which did not inhibit acetylphenylalanylpuromycin synthesis, was a strong inhibitor of peptidylpuromycin formation with native polyribosomes. Since transpeptidation was measured in both assays, the contrasting effects observed represent different interactions of the antibiotics with the discrete ribosomal states present in the two distinct assays. The reaction of puromycin with native polyribosomes is illustrated in Fig. 6. It can be seen that puromycin interacts with nascent peptides of 3 pM. As expected, one puroon polyribosomes with an apparent K:,, mycin molecule is involved in the release of each nascent peptide. Study of the kinetics of this reaction has been useful in understanding transpeptidation on polyribosomes as well as the interactions of antibiotics with ribosomes in various states.

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TABLE I11 EFFECT OF ANTIBIOTICS O N TRANSPEPTIDITION ASSAY

Antibiotic Chloramphenicol Lincomycin Carbomycin Erythromycin Niddamycin Oleandomycin Spiramycin I11 Tylosin Streptogramin A PA114A Vernamycin A A1thiomycin Sparsom ycin Amicetin Gouger0 tin Blasticidin S Anisomycin Tenuazonic acid Trichodermin Cycloheximide

Synthetic models for peptide bond synthesis"

+ + + + + + + + + + k + + + + + + +?

+d

Native peptide bond synthesisb

+

-

+ + + + + + + + +

Species inhibitedc

P P P P P P P P P P P P B B B B E E E E

a Refers to assays where NH4C1-washed ribosomes are used as well as synthetic peptidyl donors, such as AcPhe-tltNA, fMet-tItNA, polylysyl-tItNA, C-A-C-C-A(AcPhe), C-A-A-C-C-A(fMet) and others, to form peptidylpuromycin. * Refers to the synthesis of peptidylpuromycin with native peptidyl-tltNA on polyribosomcs isolated from cells. P = prokaryotic; E = eukaryotic; B = both. Inhibition; -, no effect; +, partial inhibition.

+,

In addition, the effect of antibiotics on chloramphenicol binding to 70 S ribosomes, and polyribosomes could also be used as a probe for their interaction. Since chloramphenicol inhibits both acetylphenylalanylpuromycin and peptidylpuromycin synthesis, it was expected that it would interact with both washed ribosomes and the ribosome monomers of polyribosomes. In fact, this has proved to be the case ( 2 4 ) . Those antibiotics that inhibit acetylphenylalanylpuromycin synthesis, but not peptidylpuromycin synthesis ( the niacrolides, lincosamides and 'streptogramin A antibiotics ), inhibit chloramphenicol binding to NH,Cl-washed ribosomes. However, they are essentially ineffective in inhibiting chloramphenicol binding to polyribosomes (Tables V and VI). In addition, streptogramin B antibiotics and the aminonucleosides, which inhibit

227

INHIBITORS OF PROTEIN SYNTHESIS

TABLE IV EFFECT O F AKTII~IIJTICS O N SYNTHESIS OF ACKVYLPHENYLALINYLIJUROMYCIN .\ND PEPTIDYLPUItOMYCINa

Antibiotic Chloramphenicol Lincomycin Carbomycin Erythromycin Oleandomy cin Methymycin Niddamycin Tylosin Spiramycin 111 Vernamycin A PA114A A1thiomycin Sparsomycin

Acetylphenylalanylpuromycin synthesis

Peptidylpuromycin synthesis

10-631 l0-5M l0-4hhZ lO-3M

10-oM l0-6h'I 1 0 V M l0-3M

99 98 83 104

88 88 18 103

51 60 17 134

108

23

23

1 1 94 4

1 2 114 2

100 103 102 101 104 44

103 96 112 97

105 98 91 100 92 86 95 100 98 108 96 3.5 16

64 102 114 102 103 88 103 105 99 103 110 14 1

42 92 89 10t5 96 83 94 -

98 101 1

a The data in the table are taken from Pestka (15, 80, 23) and are presented as percentage of control reactions in the absence of antibiotics.

LOG [ PUROMYCIN]

R

h XIO-5

FIG.6. Hill plot and double-reciprocal plot for puromycin participation in peptidylpuromycin synthesig. Each 50-r(.l reaction mixtures contained the components indicated in the legend to Fig. 4. Puromycin concentration was vaned, and reactions took place at 24°C for 1 minute. The interaction coefficient, n, was found to be 0.996 and K , was determined as 3.3 p h l for puromycin (log K,,, = 5.48, the intercept of the ordinate). When u = V,1,ny/2,log[o/( V,,,,, - u ) ] equals 0, and K., = 3.0 fiM (pK,,, = 5.52), as determined from the intercept of the abscissa of the Hill plot. The K,,, was determined to be 3.2 pM from the double-reciprocal plot.

chloramphenicol binding to ribosomes, are ineffective in inhibiting chloramphenicol binding to polyribosomes ( 24 ) . Furthermore, sparsomycin and althiomycin are ineffective in inhibiting chloramphenicol binding to washed ribosomes, but are strong inhibitors of chloramphenicol binding to polyribosomes (Fig. 7 ) . It can be seen that sparsomycin is a potent inhibitor of chloramphenicol binding to polyribosomes. At lo-' M,

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TABLE V EFFECT OF ANTIBIOTICS O N CHLORAMPHENICOL BINDING TO 70 S RIBOSOMES A N D POLYRIBOSOMES Antibiotic

Ribosomes Polyribosomes

+" + + +

Macrolides Lincosamides Streptogramins A Streptogramins B Aminonucleosides Sparsomycin A1thiomycin

f f

-

+

-

-

+ +

+, Strong inhibition; -, no inhibition; f,partial inhibition. ~~

~

TABLE VI EFFECTOF ANTIBIOTICSO N BINDINQ OF CHLORAMPHENICOL TO RIBOSOMES A N D TO POLYRIBOSOMEW Binding to ribosomes Antibiotic Erythromycin Carbomycin Spiramycin I11 Niddamycin Oleandomycin Methymycin Tylosin Lincomycin Celesticetin Vcrnamycin A PA114A Vernamycin Ba PA114B Sparsomycin Althiomycin C hloramphenicol Blasticidin S Gougerotin Amicetin

10-6

M 10-6 M 10-4 M 10-3 M

94 85 93 96 107 95 94 104 108 91 118 110 107 98 101 -

32 71 14 Ti8 33 .59 12 96 99 16 16 102 33 100 96 60 96 106 105

11 15 10 11 14 23 15 65 89 12 13 32 23 92 100 18 73 84 108

Binding to polyribosomes 10-6

M 10-KM10-4 M 10-3 M

99 100 81 92 go 89 94 93 101 89 100 97 94 53 98 -

-

93 87 93 87 87 66 94 94 96 93 88 95 90 20 47 54 101 102 102

78 89 92 93 74 39 92 80 87 86 90 88 93 15 22 17 97 97 114

The data in the table are taken from Pest,ka ($4) and are presented as a percentage of control reactions in the absence of antibiotics.

229

INHIBITORS OF PROTEIN SYNTHESIS

o

1.1

I

"0-7

I 10-6

I

I 10-5

I

I

0

10-4

SPARSOMYCIN [MOLARITY]

II

1.1

10-3 0-10-6

I

10-5

10-4

ALTHIOMYCIN [MOLARITY]

FIG. 7. Effect of sparsomycin and althiomycin on chloramphenicol binding to ribosomes and polyribosomes. Reactions were performed as indicated in reference 24. Antibiotic concentration is given on the abscissas. The data are plotted as a percentage of the control value for the binding of chloramphenicol to ribosomes or polyribosomes in the absence of any antibiotic. Left: This cuntrol value was 36.5 and 35.1 pmol of chloramphenicol for ribosomes [6.4 absorbancy ( A m o ) units] and polyribosomes (4.2 Amo units), respectively. Right: Control value was 45.2 and 47.0 pmol of chloramphenicol for ribosomes (6.4A, units) and polyribosomes ( 5.6 A,lo units), respectively. Symbols: chloramphenicol binding to ribosomes (0) and to polyribosomes ( 0 ) .

sparsomycin inhibition of chloramphenicol binding to ribosomes was detectable and at approximately M, inhibition of chloramphenicol binding to polyribosomes was greater than 50%. Nevertheless, at M (1000- to 10,000-fold higher sparsomycin concentrations than necessary to inhibit chloramphenicol binding to polyribosomes ), sparsomycin had little or no effect on chloramphenicol binding to washed ribosomes. The results with althiomycin were similar. Althiomycin inhibited chloramphenicol binding to polyribosomes but had little or no effect on chloramphenicol binding to washed ribosomes. These antibiotics are clear probes of the ribosomal states involved and illustrate the precise and subtle insights into the ribosomal states that they can provide. Since erythromycin inhibits transpeptidation with washed ribosomes but not with polyribosomes (3, 23), it was anticipated that this might reflect the inability of erythromycin to interact with ribosomes in polyribosomes. As shown in Table VII, erythromycin binds to ribosomes but relatively little to polyribosomes. However, if peptidyl-tRNA is removed from polyribosomes by treatment with puromycin, erythromycin binds to the resultant ribosomes. It is thus clear that the presence of peptidyl-tRNA on polyribosomes inhibits erythromycin binding and pre-

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TABLE VII BINDING OF ERYTHROMYCIN TO RIBOSOMES AND POLYRIBOSOMES BEFORE AFTER REACTION WITH PUROMYCIN'

AND

~~

Molecules of erythromycin bound/ribosome Polyribosomes or ribosomes Polyribosomes 70 S ribosomes (native) 70 S ribosomes (washed)

Plus Minus puromycin puromycin 0.17 0.82 0.75

Ratio plus: minus puromycin

0.80 0.97 0.69

4.7 1.2 0.93

,, Data taken from Pestka (84).

sumably its interaction with the ribosome (24, 24u). It should be noted that these studies were carried out with riiosomes in a static state. Ribosomes actively involved in the dynamic cycles and various states of protein synthesis, however, may, in fact, not react identically with these antibiotics. It is possible that the various conformational and translocational movements involved during elongation of the peptide chain may allow these antibiotics to interact with ribosomes on polyribosomes despite our results using a static state, that is, using polyribosomes not actively involved in protein synthesis. Nevertheless, whether or not this proves to be true for the dynamic ribosomes of protein synthesis, these agents serve as useful probes into the static ribosomal states involved.

V. Erythromycin Binding to Ribosomes Erythromycin binding to ribosomes was examined for several reasons. In the first place, valid binding constants for erythromycin binding to E . coli ribosomes has not been reported, Second, we hoped to use the erythromycin binding site as a focus for affinity labeling of ribosomal proteins and other components. Furthermore, it would be useful to correlate the effects of erythromycin derivatives on erythromycin binding to ribosomes with their antibacterial activities. Since the ribosome is the target of erythromycin action, the affinity of an erythromycin derivative for ribosomes should reflect its antibacterial activity. Any discrepancies from this correlation should, therefore, be due to alterations in other parameters, such as metabolic conversion or cellular permeability to the derivative.

231

INHIBITORS OF PROTEIN SYNTHESIS ERYTHROMYCIN MOLARITY x 10'

246810

0

PICOMOLES ERYTHROMYCINADDED TO REACTION MIXTURE

FIG.8. Binding of erythromycin to ribosomes as a function of erythromycin concentration. Each 0.50-ml reaction mixture for the filter assay, 24°C (left panel) contained 5.6 Az, units of ribosomes and other components as described. The initial concentration of ["Clerythromycin added to the reaction mixture is given on the abscissa. Dissociation ( K d = 1.0 x 10.' M ) and association (K. = 9.9 x 10' M-') constants were computed from the Scatchard plot (inset). Equilibrium dialysis, 5°C (right panel) was performed at 46 and 70 hours ( 2 5 ) . The values at 70 hours are plotted directly and as a Scatchard plot (inset). At 46 hours, the slope of the Scatchard plot was similar, but the baseline intercept was 0.91. At 70 hours, the baseline intercept was 0.86. The dissociation ( K d = 1.4 x lo-' M ) and association (K. = 7.2 x 10' M-') constants were calculated from the Scatchard plot. A computer program was used to determine the line of best fit by the method of least squares. The data for the filter assay arc presented as solid circles ( 0 ) and the data for equilibrium dialysis by open circles ( 0).

Erythromycin binding to ribosomes was examined by equilibrium dialysis as well as by the filter binding technique (Fig. 8). Th'is association constant was determined to be 7.2 x lo7 M-' and the dissociation constant 0.014 PM by equilibrium dialysis. Equilibrium dialysis was performed at 5°C. Association (K,= 9.9 x lo7 M-') and dissociation ( Kd = 0.01 PM) constants were also determined by the filter binding technique at 24°C (25). The binding of erythromycin to ribosomes is reversible (Fig. 9). The forward reaction is extremely rapid and essentially completed by the time components are mixed. The reverse reaction rate is measurable. Thus, from the equilibrium constant and the reverse reaction rate, the forward reaction rate was calculated to be 1.7 X lo7liters mol-' min-'. Equations relating the effect of erythromycin derivatives on erythro-

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20L

I

-I

MINUTES

FIG.9. Reversibility of erythromycin binding to ribosomes. Each 0.50-ml reaction mixture contained 5.6 A, units of ribosomes and other components as described in reference 25. The time course of [“Clerythromycin binding to ribosomes was followed as a function of time at 24°C. At 15 minutes, 10 ~1of a 10 mM solution of unlabeled erythromycin was added to some of the tubes as indicated (arrow) to produce a ratio of 200/1 for unlabeled to %-labeled erythromycin in these tubes. 0, No unlabeled erythromycin; 0, 0.1 pmol of unlabeled erythromycin added to each of these tubes at 15 minutes.

&UATlONS

where:

TABLE VIII RELATINQ ERYTHROMYCIN BINDING TO RIBOSOMES AND (Nu) INHIBITOR CONCENTRATION

[El = free erythromycin concentration [I] = 10 = input unlabeled antibiotic concentration [Ro] = concentration of total ribosomes potentially active in binding erythromycin [R] = concentration of free ribosomes potentially active in binding erythromycin [RE] = concentration of ribosome * erythromycin complex [RI,] = concentration of ribosome . inhibitor complex

INHIBJTORS OF PROTEIN SYNTHESIS

233

mycin binding to ribosomes are described in Table VIII. Thus, from the effect of an erythromycin derivative on erythromycin binding to ribosomes, it was possible to calculate the dissociation constant of that derivative as well as its interaction coefficient (26). This was done for approximately 50 erythromycin derivatives with the results shown in Figs. 10A and 10B. Many analogs were practically inactive in inhibiting erythromycin binding to ribosomes, and others were almost as active as erythromycin itself. When these data were plotted according to the equations presented in Table VIII, straight lines were generally obtained from which the association constants as well as the interaction coefficients for each derivative could be derived (Fig. 11). Examples of some of the erythromycin derivatives synthesized and examined are shown in Figs. 12-14. It can be seen that various portions of the erythromycin molecule can be modified with no substantial loss of binding. Specifically, the cladinose can be removed and replaced by various groups with retention of activity, and various substitutions can be made on the oxime as well as on the sugar moieties. There is a general correlation between the ribosomal binding of a derivative and its antibacterial activity (27). Analogous studies with erythromycin have been performed by others (28, 29). Similar studies were performed with leucomycin derivatives ( 3 0 ) , which complete with erythromycin binding to ribosomes as shown in Fig. 15. Binding of each leucomycin derivative to ribosomes correlated exceeding well with its antibacterial activity ( Fig. 16). With the knowledge of which sites on the erythromycin molecule could be chemically altered, a number of erythromycin derivatives with groups for covalent attachment to ribosomes have been synthesized (31 ). These erythromycin affinity analogs should help localize the erythromycin binding site on the surface of the ribosome. A fluorescent derivative of erythromycin has been prepared ( 3 1 ) for use in probing the erythromycin binding site and in fluorescence transfer measurements for determination of intraribosomal distances. Such energy transfer measurements between the bound fluorescein isothiocyanate derivative of erythromycylamine and fluorescent labeled L7 yielded a distance of approximately 70 A between these two moieties (R. Langlois, C. C. Lee, C. Cantor, R. Vince and S. Pestka, unpublished). Although the erythromycin binding site appears to bt: in the vicinity of the peptidyl portion of peptidyl-tRNA (24, 24u), further characterization and definition of the erythromycin binding site should result from affinity labeling studies with derivatives of erythromycylamine (31). After such localization, the distance measurements between the erythromycin binding site and the factordependent GTPase center involving L7/ L12 should have more relevance.

234

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B

ANTIBIOTIC MOLARITY

FIGS.10A and 10B. Effect of erythromycin analogs (see Figs. 12-14) on erythromycin binding to ribosomes. Each 0.50-ml reaction mixture contained the following components: 0.1 M KCI; 0.01 M NH,CI; 0.004 M MgCL; 0.01 M Tris-chloride, pH 7.2; 1.1 pM ["Clerythromycin; 7.5 A, units of NRCI-washed ribosomes; and erythromycin analogs at the concentrations indicated on the abscissas. Reactions were started by the addition of ribosomes and incubated at 24°C for 30 minutes. In the case of analog 28, the reaction was incubated at 24°C for 15 minutes to minimize hydrolysis of the erythromycin derivative. Assays were performed by adsorbing ribosomes to cellulose acetate-cellulose nitrate filters (26).The data are presented as a percentage of the ["C]erythromycin bound to ribosomes in the absence of any nonradioactive erythromycin or erythromycin derivatives.

VI. Effect of Chloramphenicol on the Puromycin Reaction: Models of Ribosome Function Kinetic analysis of the effect of chloramphenicol and other antibiotics on peptidylpuromycin formation stimulated some interesting concepts. The effect of sparsomycin on peptidylpuromycin synthesis is shown in Fig. 17 as a control. Sparsomycin has been reported to be a competitive inhibitor of puromycin, and its activity as a competitive inhibitor of puromycin in the synthesis of peptidylpuromycin, by analysis with a double-reciprocal plot as well as with a Dixon plot, supports the validity of this interpretation (23). The results with chloramphenicol were different. In the usual double-reciprocal plot, chloramphenicol appeared to be a mixed inhibitor of the puromycin reaction for peptidylpuromycin synthesis (Fig. 18). When a Dixon analysis was performed (Fig. 19),

INHIBITORS OF PROTEIN SYNTHESIS

-7 -6 -5 -4

-7 -6 -5 -4

-7 -6 -5 -4

-7 -6 -5 4

LOG (ANTIBIOTIC MOLARITY)

FIG.11. Graph of y as a function of antibiotic concentration for computation of association and dissociation constants. (The names of the antibiotics are given in Figs. 12-14.) The equation log { ( K O [El ( [Ro] - [RE])/[RE]) - I} = n log [I] log KI = y (Table VIII) was used to calculate KI,the association constant for the binding of inhibitor to ribosomes; KO is the association constant for the binding of erythromycin to ribosomes (9.9 x 10' M-' was used for these calculations); n is the number of inhibitor molecules binding to ribosomes; [El is the free erythromycin concentration; [Ro] is the total concentration of ribosomes potentially active in binding erythromycin; and [RE] is the concentration of the erythromycin-ribosome complex. From the data of Figs. 10A and 10B, y was plotted as a function of log [I] (26). Some representative results are presented in this figure. A computer program was used to determine the line of best fit by the method of least squares. When y = 0, n log [I] = -log Kl; thus assuming that n = 1, log KI = -log [I] at the baseline intercept. The baseline intercept was used to determine Kr. When n = 1, the KI values determined by the intercept of the abscissa or the ordinate are theoretically equivalent. KI for erythromycin was determined independently to be 9.9 x 10' M-' and, as computed above with a nonradioactive competing sample, as 1oR M-I.

+

it appeared that chloramphenicol inhibited this synthesis in two distinct phases. A fraction of the peptidylpuromycin formed was very sensitive to chloramphenicol whereas another portion was relatively resistant ( 23). This was quantitatively indicated by the Dixon plot, from which the two dissociation constants were estimated as 70 pM and 2.2 mM. Similar results were obtained with other antibiotics, such as amicetin, blasticidin S and gougerotin.

236

SIDNEY PESTKA

c.Hi'*ko+3

"0-OESOSAMINE

'"o-OESOSAMINE

*. '0-CLADINOSE

o' cn3

'0-CLAOINOSE

cn3

CHI

I

ERYTHROMYCIN A

2

...oT...o-p+::o

OESOSAMINE MODIFICATIONS OF ERYTHROMYCIN A:

-

On

0

9

-

CHI

p

0

O-

B

~

~

CHI

3

4

9c-c~~

On

N/CH3 3

~

cn3 5

C H ~ 6

FIG. 12. Structures of erythromycin and erythromycin analogs.

14

IS

17

ie

R = R'= R"= H

R = R'= R * = CH~CO-

R=H R=CH3CO-

19 R * R'. CH3CO20 R = R ' = H

FIG.13. Structures of erythromycin analogs.

237

INHIBITORS OF PROTEIN SYNTHESIS

27 28 29

30

31 32 33

34

35 36 37 38 39

40

41 42

43 44

FIG.14. Structures of erythromycin analogs.

In essence, this result does not appear to be consistent with the simple acceptor-donor site model for protein synthesis on the ribosome. The model indicates that there are two states with different sensitivities to chloramphenicol in which peptidylpuromycin can be synthesized. From the model of the donor-acceptor site hypothesis (Fig. 20), there should be only one state in which peptidyl-tRNA can react with puromycin. The chloramphenicol probe indicates that peptidyl-tRNA in fact exists in two states that can react with puromycin, one state being relatively sensitive to chloraniphenicol and the other relatively insensitive. A different model for ribosome function may be proposed to explain these results. In this model of the ribosome epicycle (Fig. 21), it is proposed that peptidyl-tRNA exists in two states or may enter the ribosome from two sides. From each side of the ribosome, the peptidyl-tRNA could enter a donor or peptidyl site, which is distinguished essentially by the end of peptidyl-tRNA. Similarly, aminoacyl-tRNA, the acceptor, would enter an acceptor area, which is reserved for the aminoacyl ter-

238

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ANTIBIOTIC MOLARITY

FIG.15. Effect of leucomycin ( L M ) analogs on erythromycin binding to ribosomes. Experiments were performed as described in the legend to Figs. 10A and 10B.The data are taken from Pestka et al. (30).

minus of aminoacyl-tRNA. Nevertheless, both peptidyl- and aminoacyltRNA could enter these sites from either side. Hence, it is postulated that there may be two sites for chloramphenicol binding to ribosomes and these may affect peptidyl transfer from the two directions with different sensitivities. Alternatively, there may be one site for chloramphenicol binding that affects the peptidyl transfer from the two sides differentially. Equilibrium dialysis with NHaC1-washed ribosomes, in fact, indicates that there may be two sites for chloramphenicol binding, one with high affinity and one with low affinity for ribosomes (32).

VII. The Sistrand: the Translational Unit It has been known for some time that polycistronic messenger RNAs exist in bacteria. The mRNA transcribed from the lactose and histidine operons of Escherichia coli (33j 34) as well as from the RNA phages f2, R17, MS2 and Qp (35-39) represent such polyci,stronic messages.

239

INHIBITORS OF PROTEIN SYNTHESIS

1.5

-

1.0 -

-

6.0

5.5

50

4.5

4.0

3.5

FIG.16. Minimal inhibitory concentration (MIC) as a function of concentration for 50% inhibition of erythromycin binding to ribosomes for leucomycins and leucomycin derivatives. The log of the MIC (rg/ml) for each of the compounds as determined against Staphylococcus aureus is plotted as a function of the p L % for these same compounds numbered as follows: 1, leucomycin A;2, leucornycin A,; 3, leucomycin A,; 4, leucomycin A; 5, leucomycin As; 6, magnamycin B; 7, leucomycin &; 8, leucomycin U; 9, leucomycin A3 N-oxide; 10, demycarosyl leucomycin As; 11, 9-dehydro-18-dihydroleucomycin A3; 13, 2’-O-acetyl-3’-desdimethylamino-3’-oxoleucomycin A,. The line of best fit was determined by the method of least squares with a computer program. For determination of this line of best fit, only the filled circles were used. This line is described by the following equation: log (MIC) = 6.861 - 1.362 PI&%.Thus, for any pIGos, value the estimated MIC can be calculated. Data are from Pestka et al. ( 3 0 ) .

These RNA phage messages carry the information for three different cistrons and they are translated into three separate polypeptides. Poliovirus RNA is also polycistronic, leading to the production of a number of different proteins (40,41).However, the mechanism by which each of these mRNAs is translated is quite different. The mRNA for the RNA phages has three separate initiation and termination sites separated by nontranslated regions. Each peptide is produced separately from start to finish. On the other hand, the polio RNA has a single initiation and single termination site. During translation a single large precursor, which is subsequently cleaved into the individual virus proteins, is synthesized (40,41 ). The term “polycistronic” in no way distinguishes between these two mechanisms of translation and is a misnomer when applied to translation of mRNA since it is a genetic term. At present, there is no term describing the translational unit. We have proposed the term “sistrand,” derived from “single initiation single termination strand,’’ for

240

SIDNEY PESTKA

PUROMYCIN MOLARITY

do6

11s

10-5

FIG.17. Kinetics of sparsomycin inhibition of peptidylpuromycin formation. Peptidylpuromycin synthesis was determined at the various concentrations of sparsomycin. Reaction conditions are similar to those described in the legend to Fig. 4. 0, NO sparsomycin; 0, 1 pM sparsomycin; A, 3 gM sparsomycin. A reciprocal plot of the data of the left panel ( VmSx= 4.2) is presented in the right panel (puromycin, K,,,= 2.4 pM; sparsomycin, K I = 0.16 pM).

5 4

3 2 I

0

5

K

)

l

5

2

0

2

[3H]PUROMYClN MOURITY x106

5

-

2

0

2

4

11s

6

8

K

)

10-5

FIG. 18. Kinetics of chloramphenicol inhibition of peptidylpuromycin formation. Reaction conditions were similar to those of Fig. 4 (23 ), The concentration of puromycin was vaned as shown on the abscissa. A reciprocal plot of the data of the left panel is presented in the right panel. 0, No chloramphenicol; A, 0.1 mM chloramphenicol; 0, 0.5 mM chloramphenicol; A, 1 mM chloramphenicol.

the unit of translation ( 4 2 ) . The sistrand represents the translational unit whereas the cistron represents the genetic unit. The sistrand is that unit of mRNA that lies between an initiation and termination signal (Fig. 22). Thus, these phage mRNAs are polysistrandic, having three

INHIBITORS OF PROTEIN' SYNTHESIS

Ki2

Ki1

CHLORAMPHENICOLMOURITY

~ 0 4

I

FIG.19. Kinetics of chloramphenicol inhibition of peptidylpuromycin formation. Reaction conditions were similar to those of Fig. 4 (23).Chloramphenicol concentration was vaned as indicated on the abscissa at four different puromycin concentrations. Polynbosomes were added last to start the reactions. The data of the left panel are plotted and calculated in the right panel ( Ktl = 70 PM; Kt, = 2.2 mM) ac13.7 pM puromycin; A, 4.55 pM purocording to Dixon [BJ 55, 170 (1953)l. mycin; 0, 2.28 p M puromycin; A,1.14 p M puromycin.

e,

distinct initiation and termination sites, as well as polycistronic. Polio mRNA is monosistrandic, but polycistronic. For some time, it has been suspected that most, if not all, eukaryotic mRNAs are monosistrandic (40,43, 44), but the evidence was not definitive. With the use of specific inhibitors of initiation, it should be possible to block protein synthesis at initiation, and thus functionally fix ribosomes at the initiation sites of mRNA. Therefore, mRNAs with a single initiation site should appear in the monoribosome region, those with two in the

n

- CODON

PEP

TRANSPEP-

RECOGNITION

TIDATION

EC -

t

-

e

TRANSLOCATION

FIG. 20. Schematic illustration of the donor ( P ) and acceptor ( A ) site model of ribosome function.

CODON

RECOGNITION

-

c

= I

b

TRANSLOCATION

n

- TRANSEP-

CODON

TIDATION

RECOGNITION

f

e

d

FIG.21. The ribosome epicycle; a model of ribosome sites and ribosome function for codon recognition, transpeptidation and translocation. Major features of the model are as follows: ( a ) There are two or more sites for tRNA binding on the 50 S subunit. Although the model would be essentially similar for any number of sites greater than two, the minimum number of sites on the 50 S subunit is two. ( b ) The two sites on the 50 S subunit are functionally similar but not identical. Transpeptidation can occur in both directions as illustrated ( b , e ) . As a consequence of this, each site can contain aminoacykRNA, peptidyl-tRN.4 or deacylated tRNA. The direction of transpeptidation depends on the nature of the constituents of the sites rather than on the sites themselves. ( c ) There is only one site for tRNA binding on the 30 S subunit; this is the decoding site. By a conformational change (shown in the figure) or by a rotation of the subunit, the site can align with either 50 S site. Because of this separate relative movement over many angstroms, the subunits should be separate particles. ( d ) Translocation involves movement of mRNA along the 30 S subunit, realignment of the 30 S subunit decoding site with the second 50 S site, and removal of deacylated tRNA. It is probable that removal of tRNA is the step dependent on factor EF-G and GTP; realignment of the 30 S site with the second 50 S site may or may not be dependent on EF-G. It is possible that factor EF-Tu may have a role here. ( e ) In order for the active center of the peptidyltransferase to interact in identical ways with peptidyl-tRNA and aminoacyl-tRNA in the two sites, it may be necessary that the active site of the peptidyltransferase rotate with respect to the sites. However, since the C-C-A( peptidyl ) end of peptidyl-tRNA does not bind firmly to ribosomes, it may be possible for the 3’-terminal portion of peptidyl-tRNA to align identically with the catalytic center of the peptidyltransferase without rotation of the catalytic center. This may be possible because of free rotation around each of the bonds of the C C - A ( peptidyl) end. Analogously, although the C-C-A( amino acid) of aminoacyl-tRNA is the fixed moiety, the free rotation around each bond of this end may place the C-C-A(amino acid) from either 50 S site in the same stereochemical position with respect to the peptidyltransferase.

243

INHIBITORS OF PROTEIN SYNTHESIS INITIATION

TERMINATION

v

v m

v m

1 SISTRAND

( PE::iRgk 3 SISTRANDS

(POLYCISTRONIC)

FIG.22. Schematic illustration of the sistrand, the translational unit; strand of mRNA between initiation and termination signal.

diribosome region, those with three in the triribosome region, and so forth. Although the usual initiation inhibitors were not sufficiently specific to allow performing such studies, we found a group of inhibitors that were useful ( 4 5 ) . Studies with the harringtonine alkaloids indicated that these agents are specific inhibitors of initiation in intact HeLa cells and that most ribosomes appear in the monosome region in cells exposed to harringtonine ( 4 5 ) . These results indicate that HeLa cell mRNAs may be monosistrandic. It is likely that many cellular messages from animal cells are polycistronic, but monosistrandic. It is of interest to ask why eukaryotic cells should translate mRNAs predominantly via monosistrandic messages and to consider what advantages, if any, such posttranslational controls offer. Since prokaryotes are essentially nutritionally oriented with minimal differentiation, the synthesis of proteins essentially as final products corresponds to their immediate needs, i.e., their proteins are synthesized and immediately utilized. Differentiation, in contrast, requires, and by definition means, that cells evolve specialized functions. A cell in one part of the organism synthesizes what a cell or area in another location requires. In other words, protein products from one part of the system may be required in another part of the system and thus must be transported through space and time within the system. A further requirement may be that the product be present in an inactive form until needed. A priori, there appears to be no reason why prokaryotic polysistrandic mRNA cannot accomplish this. However, if eukaryotes routinely synthesized precursor proteins that are inactive and that require activation prior to function, eukaryotes would have a built-in mechanism for differentiation of cellular functions. The blood coagulation system provides a good example of the synthesis of numerous protein precursors in the liver. These proteins are transported to a particular place (the bloodstream) and normally function only when required (bleeding), at which time they are activated. It is possible that the synthesis of all proteins as inactive precursors provides a mechanism for the evolutionary beginnings of cellular differentiation.

244

SIDNEY PESTKA

It appears that inhibitors of protein synthesis and ribosome function allow us a striking insight into ribosome function and soon, perhaps, into ribosome structure. Our knowledge of protein synthesis has allowed us to delineate the mode of action of many of these agents. The agents themselves have allowed us insights and views into ribosomes and protein synthesis not available by other means.

REFERENCES I. J, Lucas-Lenard and F. Lipmann, ARB 40,409 ( 1971 ).

2. R. Haselkorn and L. B. Rothman-Denes, ARB 42,397 ( 1973). 3. S. Pestka, Annu. Rev. Microbiol. 25,487 ( 1971). 4. B. Weisblum and J. Davies, Bacteriol. Reo. 32, 493 (1968). 5. S. Pestka, in “Methods in Enzymology” Vol. 30: Nucleic Acids and Protein Synthesis (L. Grossman and K. Moldave, eds.), Part F, p. 261. Academic Press, New York, 1974. 6. N. R. Towers, H. Dixon, G. M. Kellerman and A. W. Linnane, ABB 151. 361 (1972). 7. D. Vazquez, FEBS Lett. 40, S63 ( 1974). 8. R. E. Monro and K. A. Marcker, J M B 25,347 ( 1967). 9. S. Fahnestock, H. Neumann, V. Shashoua and A. Rich, Bchem 9,2477 (1970). 10. S. Pestka, R. Vince, S. Daluge, and R. Harris, Antimicrob. Ag. Chemother. 4, 37 (1973). 11. S. Pestka, PNAS 89, 624 ( 1972). 12. S . Pestka, in “Methods in Enzymology” Vol. 30: Nucleic Acids and Protein Synthesis (L. Grossman and K. Moldave, eds. ), Part F, p. 462. Academic Press, New York, 1974. 13. L. P. Gavrilova and A. S. Spirin, in “Methods in Enzymology” Vol. 30: Nucleic Acids and Protein Synthesis (L. Grossman and K. Moldave, eds.), Part F, p. 452. Academic Press, New York, 1974. 14. S. Pestka, BBRC 40, 667 (1970). 15. S. Pestka and N. Brot, JBC 248,7715 (1971). 16. S. Pestka, JBC 244, 1533 (1969). 17. S. Pestka and J. W. Bodley, in “Antibiotics” (J. W. Corcoran and F. E. Hahn, eds.), Vol. 111, pp. 551-573. Springer-Verlag, Berlin and New York, 1974. 18. S. Pestka, in “Antibiotics” (J. W. Corcoran and F. E. Hahn, eds.), Vol. 111, pp. 480-486. Springer-Verlag, Berlin and New York, 1974. 19. S. Pestka, Abst. Papers, 166th Nut. Meet. Amer. Chem. SOC., Chicago, Illinois, 1973. Carb #2. 20. S . Pestka, ABB 138, 80 (1970). 21. H. Fujimoto, T. Kinoshita, H. Suzuki and H. Umezawa, J . Antfbtot. 23, 271 (1970). 22. S. Pestka, in “Antibiotics” (J. W. Corcoran and F. E. Hahn, eds.), Vol. 111, pp. 323-326. Springer-Verlag, New York, 1974. 23. S. Pestka, JBC 247, 4669 (1972). 24. S. Pestka, Antimicrob. Ag. Chemother. 5, 255 (1974). 24a. N. L. Oleinick and J. W. Corcoran, Roc. Int. Congr. Chemother., 6th, 1969 p. 202 ( 1970). 25. S. Pestka, Antimicrob. Ag. Chemother. 8, 474 (1974).

INHIBITORS OF PROTEIN SYNTHESIS

245

25a. R. Harris and S. Pestka, JBC 248, 1168 (1973). 26. S. Pestka and R. A. LeMahieu, Antimicrob. Ag. Chemother. 6, 479 (1974). 27. S. Pestka, R. A. LeMahieu and P. Miller, Antimicrob. Ag. Chemother. 6, 489 (1974). 28. J. A. Wilhelm, N. L. Oleinick and J. W. Corcoran, Antimicrob. Ag. Chemother.1967 p. 236 (1968). 29. J. C.-H. Mao and M. Putterman, JMB 44,347 (1969). 30. S. Pestka, A. Nakagawa and S. bmura, Anttmicrob. Ag. Chemother. 6, 606 (1974). 31. R. Vince, D. Weiss and S. Pestka, Antimicrob. Ag. Chemother. 9, 131 (1976). 32. J. L. Lessard and S. Pestka, JBC 247, 6909 ( 1972). 33. Y. Kiho and A. Rich, PNAS 54, 1751 (1965). 34. R. G. Martin, CSHSQB 28, 357 (1963). 35. D. Nathans, M. P. Oeschger, K. Eggen and Y. Shimura, PNAS 56, 1844 (1966). 36. E. Viiiuela, I. D. Algranati and S. Ochoa, E J B 1, 1 (1967). 37. H. F. Lodish and H. D. Robertson, CSHSQB 34,655 (1969). 38. J. Argetsinger Steitz, CSHSQB 34, 621 (1969). 39. H. D. Voonna, FEBS Abstr. 6, 114 (1969). 40. D. Baltimore, M. F. Jacobson, J. Asso, and A. Huang, CSHSQB 34,741 (1969). 41. J. L. Saborio, S. S. Pong and G. Kocli, J M B 85, 195 ( 1974). 42. J. S. Tscherne and S. Pestka, Amer. SOC. MicroMol. Abstr., 75th Annu. Meet. K105, p. 164 (1975). 43. E. L. Kuff and N. E. Roberts, J M B 26,211 (1967). 44. N. S. Petersen and C. S. McLaughlin, J M B 81,33 (1973). 45. J. S. Tscherne and S . Pestka, Antimicrob. Ag. Chemother. 8,479 (1975).