Eur. J. Biochem. 68, 355- 364 (1976)

Initiation of the Polypeptide Chain by Reticulocyte Cell-Free Systems Survey of Different Inhibitors of Translation

Manuel FRESNO, Luis CARRASCO, and David VAZQUEZ

Instituto de Bioquimica de MacromolCculas, Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas and Universidad Autonoma de Madrid

(Received April 12/July 7, 1976)

In order to elucidate the mechanism of action of inhibitors that block the initiation of protein synthesis in mammalian systems, we have studied the following steps: (a) formation of the ternary complex Met-tRNA, . IF-E, . GTP, (b) binding of the initiator Met-tRNA, to the 40-S ribosomal subunit in the presence of initiation factors and dependent or not on the addition of mRNA, (c) for- mation of the initiation complex with 80-5 ribosomes and (d) formation of the first peptide bond.

Adrenochrome, aurintricarboxylic acid, polydextran sulphate, pyrochatechol violet and show- domycin block the formation of the ternary complex Met-tRNA, . IF-E, . GTP. Edeine Al , aurin- tricarboxylic acid and polydextran sulphate block the binding of the mRNA to the 40-S ribosomal subunit. Pactamycin induces the formation of stable smaller initiation complexes which are unable to go through the subsequent steps of initiation. Stimulation of the binding of the initiator Met-tRNA, to the 80-S ribosome in the presence of initiation factors is observed with sparsomycin and anti- biotics of the sesquiterpene family (verrucarin A, trichodermin and trichothecin). However, these antibiotics block the reaction of the bound Met-tRNAf with puromycin. Narciclasine has no effect on the binding of the initiator to the ribosome but strongly blocks its reaction with puromycin.

We have developed a simple technique to detect the Met-tRNA, . 40-S-subunit . poly(A,G,U) initiation complexes by chromatography on Sepharose 6B columns. The requirements for the forma- tion of such complexes measured by this technique and its comparison with the sucrose gradient centrifugation method are described.

The early steps of translation in eukaryotic cells are not yet well resolved although the general scheme of the process has been partially elucidated (for re- view see [l]). Thus it has been established that Met-tRNA,, the initiator substrate in eukaryotic cells (for review see [2]) binds to the 40-S ribosome subunit in the presence of GTP and the initiation factor IF-E, forming the complex Met-tRNAf . IF-E, . GTP '40-S- subunit; mRNA binds to this complex in a further reaction in which the initiation factor IF-E3 is required [3-51. The 60-S ribosome subunit joins the Met- tRNA, . 40-S-subunit . mRNA initiation complex in a reaction requiring cooperative interaction of the in- itiation factors (IF-El, IF-E, and IF-E5) and probably ATP, finally yielding the Met-tRNA, . 80-S-ribosome . mRNA initiation complex with the initiator bound to the P-ribosomal site (for review see [6]).

I t is well known that some translation inhibitors specifically act on the initiation phase but the precise step blocked by the inhibitor is not yet known in all cases (for review see [7]). Furthermore evidence con-

Note. The nomenclature of Staehelin et al. [6] for the initiation factors from mammalian cells (IF-E1, IF-E2, IF-E3, IF-E,, IF-E5 and IF-E6) is adopted in this paper.

cerning the effects of the inhibitors was obtained in some cases in different types of cells and cell-free systems. Therefore we have resolved the different steps of initiation to study systematically in model systems from rabbit reticulocytes the effects of a num- ber of translation inhibitors. The results obtained are presented in this paper.

MATERIALS AND METHODS

Preparation of' Ribosomes and Ribosomal Subunits

Rabbit reticulocyte ribosomes and ribosomal subunits were prepared essentially as already des- cribed [S]. Rabbits were made anaemic by daily in- jection of phenylhydrazine. The reticulocytes were collected by centrifugation and washed three times with a solution containing 140 mM NaCl, 5 mM KCl and 5 mM MgCl,. Reticulocytes were lysed by ad- dition of 2 vol. of 4 mM MgC1, and 1 mM 2-mer- captoethanol followed by strong shaking of the sus- pension for 1 min; 0.2 vol. of 1.5 M NaCl was finally added. The lysate was centrifuged at 15000xg for 20 min to pellet cells and cell debris which were dis- carded. The supernatant was centrifuged again at

356 Inhibitors of Polypeptide Initiation in Eukaryotic Systems

130000xg for 2 h. The pellet was resuspended in 50 mM Tris-HC1 buffer, pH 7.4, 0.2 mM ethylene- diamine-tetraacetate potassium salt, I mM dithio- threitol, 5 mM MgCI, and 250 mM sucrose. 4 M KCI was added by drops with slow stirring at 0 "C until the final concentration of KCl was 0.5 M and the stirring was continued for 10 min more. The suspen- sion was centrifuged at 130000 x g for 2 h and the resulting supernatant will be referred to as the ribo- somal wash fraction.

The pellet was resuspended in 50 mM Tris-HC1 buffer, pH 7.4, 0.5 M KCI, 10 mM MgCl,, 7 mM 2- mercaptoethanol and sedimented by centrifugation overnight at 105000 x g through a discontinuous sucrose gradient (20 - 60%) in the same buffers. The pellets were resuspended in 50 mM Tris-HC1 buffer, pH 7.4, 5 mM MgCl,, 25 mM KCl, 1 mM dithio- threitol and stored in small aliquots in liquid nitrogen.

Preparation of Initiation Factors

The ribosomal wash fraction was precipitated with 30- 70% ammonium sulphate, the precipitate was resuspended in 50 mM Tris-HCl buffer, pH 7.4, 0.2 mM ethylene-diamine-tetraacetate potassium salt, 1 mM dithiothreitol, 250 mM sucrose and dialyzed for 2 h against the same buffer. This suspension was used in some experiments and is referred to as crude initiation factors; for further purification 1 ml of this suspension (about 30 mg protein/ml) was applied to a DEAE-cellulose column (10 x 1 cm) equilibrated with the same buffer containing 4 mM KCl. The frac- tion that was not retained in the column was dis- carded and the same buffer described above but con- taining 0.15 M KCI was then applied to the column. The protein fraction eluted has a Met-tRNA, binding activity both in the presence and in the absence of ribo- somes. The column was further washed with the above buffer but containing 0.4 M KC1 and the protein fraction eluted has little Met-tRNA, binding activity.

The fractions eluted at 0.15 M KCl with Met- tRNA, binding activity were pooled and are referred to as the crude initiation factor IF-E,. For further purification, when required, it was applied to a Sepha- dex G-200 column (30 x 4 cm) equilibrated with 50 mM Tris-HC1 buffer, pH 7.4, 50 mM KCI, 1 mM di- thiothreitol, 0.2 mM ethylene-diamine-tetraacetate potassium salt and 250 mM sucrose. The fractions eluted were tested for Met-tRNA, binding; the frac- tions with the highest activity were pooled, stored in small aliquots in liquid nitrogen and will be referred to in this work as initiator factor IF-E,.

Preparation of r3' SjMPt-t RNA,

Yeast tRNA (General Biochemicals) was charged with [35S]methionine (50 Ci/mmol) by using a crude

preparation of aminoacyl-tRNA synthetases. This was prepared from the supernatant S-100 fraction of Escherichia coli (resulting by sedimentation of the ribo- somes at 100000 x g [S]) by protamine sulphate precip- itation, to eliminate endogenous tRNA, and further precipitation of the aminoacyl-tRNA synthetases from the supernatant fraction with 30 - 70% ammonium sulphate. [3'S]Met-tRNAf was separated from pro- teins by phenol extraction and precipitated with etha- nol at -20 "C. It was further purified by Sephadex G-25 and benzoylated DEAE-cellulose column chro- matography.

Met-tRNAs . IF-E, ' GTP Complex Formation

Formation of the Met-tRNA,. IF-Ez . GTP com- plex was determined by retention on nitrocellulose filters. Reaction mixtures of 0.1 ml contained 50 mM Tris-HCI buffer, pH 7.4, 100 mM KCl, 8 mM 2-mer- captoethanol, 0.2 mM GTP, 0.16 pmol [35S]Met- tRNA,, initiation factor IF-E, and other components as indicated under figure and table legends. Incubation was for 20 min at 37 "C and the reaction was stopped by addition of 2 ml cold buffer (20 mM Tris-HC1 buffer, pH 7.4, 60 mM KCl) and immediately filtered. Filters were dried and radioactivity was determined in a scintillator spectrometer.

Ribosome Initiation Complexes

Sepharose 6B column chromatography was mainly used to detect the ribosome initiation complexes : either Met-tRNA, . IF-E, . GTP . 40-S-subunit or Met-tRNAf . 80-S-ribosome . poly(A,G,U). Other workers evidence suggests that initiation factors might be released when this final complex is formed on the ribosome 191. Reaction mixtures of 0.2 ml contained 50 mM Tris-HCl buffer, pH 7.4, 100 mM KCl, 3 mM MgCl,, 8 mM 2-mercaptoethanol, 0.2 mM GTP, 0.32 pmol t3%]Met-tRNA,, 3 pg poly(A,G,U), either 40 pg IF-E2 or 120 pg protein crude initiation factors preparation and either 0.6 A260 unit 40-S ribosomal subunits or 0.8 unit ribosomes. Incubation was for 20 min at 37 "C and the mixtures were immediately applied to a Sepharose 6B column (15 x 1 cm) equi- librated with 50 mM Tris-HC1 buffer, pH 7.4, 60 mM KCl, 6 mM MgCI, and 10 mM 2-mercaptoethanol. 125-p1 fractions were collected and the radioactivity was estimated in a scintillation spectrometer.

Ribosome initiation complexes were also detected by centrifugation in sucrose density gradients. Reac- tion mixtures prepared as described above were layered over 5-ml linear sucrose gradients (1 5 - 30%) in 50 mM Tris-HCI buffer, pH 7.4, 60 mM KC1 and 5 mM MgCl,. Centrifugation was at 0 "C for 80 min at 48000 rev./min in an SW-50.1 rotor. Gradients were monitored at 254 nm in an Isco fractionator. 125-p1

M. Fresno, L. Carrasco. and D. Vazquez 357

fractions were collected and the radioactivity was determined in a scintillation spectrometer.

(3s SJMethion~l-puromycin Synthesis

The Met-tRNAf . 80-S-ri'bosome . poly(A,G,U) complex was formed in two sequential steps. In the first one 80-pl reaction mixtures containing 50 mM Tris- HC1 buffer, pH 7.4, 100 mM KCI, 8 mM 2-mercapto- ethanol, 0.2 mM GTP, 0.16 pmol [3sS]Met-tRNA, and either 20 pg crude IF-E2 or 60 pg protein crude initiation factors were incubated at 37 "C for 10 min. 10 pl of a mixture containing 30 mM MgCI,, 0.4 unit ribosomes and 3 pg poly(A,G,U) were then added and incubation continued for 10 min at 37 "C. For the puromycin reaction, 10 pl of 10 mM puromycin were added to the above mixture and incubated for 10 min at 37 T. The reaction was stopped by addition of 0.9 ml cold 1.0 M potassium phosphate buffer, pH 8.0, and 1 ml ethyl acetate was finally added to extract by vigorous mixing the [3s Slmethionyl-puromycin formed [lo]. 0.75 ml of the ethyl acetate phase was taken and mixed with 2 ml Bray's solution to estimate radioactivity in a scintillation spectrometer.

RESULTS

Kinetics and Inhibition of Met-tRNA, ' IF-E, . GTP Fornzation

Formation of the Met-tRNA, . IF-E, . GTP coin- plex was detected by Sephadex G-100 column chroma- tography and by retention in nitrocellulose filters [ll , 121. The complex is very rapidly formed at either 37 "C (Fig. 1A) or 0 "C (not shown). Optimal con- centrations of K + are very similar to those required for protein synthesis (Fig. IB), whereas a lower con- centration of M$+ (1 mM) is required for optimal

0.100,

01, 1'0 2'0 ;o 410 Time (min)

Table 1 . EJf'ect of inhibitors on the formution .f ' the ternury cornple.u (35S]Met-tRNAf . IF-E, ' GTP The formation of the ternary complex was carried out in 0.1-mI reaction mixtures which contained 50 m M Tris-HCI buffer, pH 7.4, 100 m M KC1, 0.5 m M MgCI2, 8 m M 2-mercdptoethano~, 0.2 mM GTP, 0.16 pmol [35S]Met-tRNA, and 20 pg of purified IF-E,. The mixtures were incubated for 20 min at 37 "C. At the end of the in- cubation period, 2 ml of cold 20 mM Tris-HC1 buffer, pH 7.4, con- taining 60 mM KCI was added to stop the reaction, the mixtures were filtered through nitrocellulose fillers and the radioactivity estimated in a liquid scintillation spectrometer. [3sS]Mct-tRNAl binding in the control was 0.040 pmol

Inhibitor added Concentration [%]Met-tRNAf bound

mM control

A urintricarboxylic acid 0.1 7 Adrenochrome 0.1 31 Polydextran sulphate 0.001 0 Pyrochatechol violet 0.1 1 N-Ethylmaleimide 10 11 Showdomycin 1 .o 25 Chartreusin 1 .o 99 Pactamycin 0.1 87 Edeine A, 0.01 1 04 2-(4-Methyl-2,6 dinitro- ani1ino)-N-methyl- propionarnide 0.1 94 Tetracycline 1 .o 87 Cycloheximide 0.1 98

formation of the complex (Fig. 1 B) as other workers have observed [12- 141.

Adrenochrome, aurintricarboxylic acid, poly- dextran sulphate, pyrocatechol violet, N-ethylmaleimi- de and showdomycin are very effective inhibitors of Met-tRNA, . IF-E2 . GTP complex formation whereas' pactamycin, cycloheximide and edeine Al have no effect (Table 1). The inhibitory effect of aurintricarb- oxylic acid has previously been observed [15].

0.100

- - 0.080

Q 1

D c

li 2 0.060 4' z 0.040

c

i2 2 0.020 - 0

0

Fig. 1. Ternury conzplex formution. (A) Time course of ternary complex formation: 0.1-ml reaction mixtures contained 50 mM Tris-HCI buffer, pH 7.4, 100 m M KCI, 0.5 mM MgCI,, 8 mM 2-mercaptoethanol, 0.2 mM GTP, 0.16 pmol [35S]Met-tRNA, and 25 pg purified IF-E,. The incubation was at 37 "C for the indicated time and the reaction was stopped and processed as described in Methods. (B) Effect of ionic conditions on the ternary complex formation : conditions were essentially a s described in (A). Effect of M$+ concentration (-0) and K +

concentration (O----O)

358 Inhibitors of Polypeptide Initiation in Eukaryotic Systems

,121 I I I

H i'i I i

u - ~ 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 50 Fraction number

Fig. 2. Isolation of 40-S-subunit . [35S]Met-tRNA, complex by Sepharose 6B column chromatography in presence of crude initiation factors. Conditions were as described in Methods. (A) Complete system; (B) minus crude initiation factors; (C) minus poly(A,G,U); (D) plus 0.1 mM pactamycin; (E) plus 0.01 mM edeine A,; (F) plus 1 mM chartreusin; (G) plus 0.1 mM aurintricarboxylic acid; (H) initiation factors were preincubated with 10 mM N-ethylmaleimide for 10 min at 37 "C and the excess of inhibitor was neutralized with 2-mercapto- ethanol, before initiation of the reaction; (I) 4 0 3 ribosome subunits replaced by 3 0 3 ribosome subunits from Escherichin coli

Binding of Met-tRNA, to the 4 0 3 Ribosomal Subunit

The ternary complex Met-tRNAf . IF-E, . GTP binds to the 40-S ribosomal subunit in a reaction which does not require mRNA if the initiation factor IF-E, is present in the assay [3-51. We have studied this reaction initially in the presence of a crude prep- aration of initiation factors detecting the complex using Sepharose 6B columns since it is easily separated in the void volume (Fig. 2). Our crude preparation of initiation factors appears to contain not only IF-E, but also IF-E, since the complex is formed even in the absence of mRNA (Fig.2). Edeine A,, pactamycin, aurintricarboxylic acid, chartreusin and tetracycline

do not inhibit this reaction. However, formation of the complex is inhibited by pretreatment of the ini- tiation factors with N-ethylmaleimide (Fig. 2 H) sug- gesting that the -SH groups of the factors are re- quired [14,16]. However pretreatment of the 40-S subunits with N-ethylmaleimide only slightly inhibits the reaction (results not shown). No complex is formed with E. coli 30-S ribosomal subunits (Fig. 21).

We also developed a system to study Met-tRNA, binding in the presence of mRNA using a partially purified preparation of IF-E, devoid of IF-E, activity (Fig. 3). The complex formed was detected by Sepha- rose 6B columns. Formation of this Met-tRNA, . 40- S-subunit . poly(A,G,U) complex requires GTP which

M. Fresno, L. Carrasco, and D. Vazquez 359

A

b L G

L 10 20 30 40

B

E &!! F

L I

L 10 20 30 40

Fig. 3. Isolation of 40-S-subunit . [35S]Met-tRNA, complex by Sepharose 6B column chromatography in presence ojpurified IF-E, initiation factor. Conditions were as described in Methods. (A) Complete system; (€3) minus IF-E,; (C) minus poly(A,G,U); (D) GTP replaced by 0.2 mM Guo-Pz-CH2-P; (E) plus 0.1 mM aurintricarboxylic acid; (F) plus 1 pM polydextran sulphate; (G) plus 10 pM edeine A1 ; (H) plus 0.1 mM pactamycin; (I) plus 1 mM chartreusin

can be replaced by Guo-P2-CH2-P but not by ATP (not shown). Edeine A,, aurintricarboxylic acid and polydextran sulphate are effective inhibitors of the reaction whereas chartreusin has only a small in- hibitory effect and pactamycin slightly enhances the reaction (Fig. 3) .

Complex formation with either a crude preparation of initiation factors containing IF-E2 and IF-E3 acti- vities or a purified preparation of IF-E2 (deprived of IF-E3 activity) and poly(A,G,U) was also detected by sucrose centrifugation (Fig. 4). The stimulatory effect exerted by pactamycin in this assay in the presence of crude initiation factors and poly(A,G,U) is remark- able.

Binding of Met-tRNA to 80-S Ribosomes

We have studied this reaction by using a system containing initiation factors, GTP, ribosomes and Met-tRNAf (Fig. 5) apparently confirming that the complex can be formed in the absence of poly(A,G,U) [17]. Complex formation is also observed when reti- culocyte ribosomes are replaced by yeast ribosomes. Sepharose 6B columns cannot separate the complex with 80-S ribosomes from that with 40-S ribosome subunits. However we have resolved two peaks of radioactivity by centrifugation, one that sediments faster corresponding to the complex formed on the 80-S ribosomes and the slower-sedimenting one COT-

360 Inhibitors of Polypeptide Initiation in Eukaryotic Systems

10 30 2 0 10

1.5

1.4

1.3

3.2

3.1 * LD N

r m

c m n

0 L

3.4 4"

3.3

3.2

3.1

0

Fraction number

Fig. 4. Analysis uf 40-S-suhunii . [35S]Met - tRNAJ cornplrv by SUCI'OSE gradient centrifugation. Conditions were as described in Methods. (A) With 120 pg of crude initiation factors; (B) with 40 pg of purified IF-E, initiation factor; (C) without initiation factors; (D) as in (A) plus 0.1 mM pactamycin. (~----o) [35S]Met-tRNAf bound; (-) absorbance at 254 mM

responding to the complex formed on the 40-S ribo- some subunits (Fig. 6A). An enhancement in the formation of the initiation complex on the ribosome is observed in the presence of verrucarin A (Fig. 6 D ; 1 9 2 x of the control), trichodermin (Fig.6E; 143% of the control), trichothecin (Fig.6F; 120% of the control) and sparsomycin (Fig.6G; 185% of the con- trol). This enhancement of the initiation complex is not observed in the 40-S ribosomal subunit (Fig. 6D, E, F,G). On the other hand pactamycin stimulates formation of the smaller initiation complex (Fig. 6H) in agreement with the results shown in Fig.4. Pre- treatment of the ribosomes with N-ethylmaleimide has no effect on the reaction (Fig. 61).

When initiation factors were omitted no dissocia- tion of ribosomes took place and therefore the presence of 40-S subunits was not observed (Fig. 6C).

Formation of the First Peptide Bond In the above system using 80-S ribosomes a func-

tional initiation complex SO-S-ribosome . Met-tRNA, . mRNA is formed with the Met-tRNA, bound to the

donor site since it is reactive with puromycin and [35S]methionyl-puromycin is synthesized (Table2). An initiation complex (Fig. 7A) similarly reactive with puromycin (Fig. 7B) was also obtained using 80-S ribosomes and our crude IF-E, preparation after DEAE-cellulose purification (crude IF-E2). The coin- plex is also formed with 40-S subunits (Fig.7A) but it is not reactive with puromycin as expected (Fig. 7B). Therefore it is likely that our preparation of crude IF-E, still contains the other non-ribosomal protein( s) or factor(s) required for the joining of the 60-S ribo- somal subunit to the smaller initiation complex [6, 17 -201. Essentially similar results were obtained '

when the effects of a number of translation inhibitors were tested on peptide bond formation by initiation complexes obtained with 80-S ribosomes using either crude IF-E, or the preparation of crude initiation factors. However, different results were obtained with pactamycin (Table2) suggesting that a protein pre- sent in the preparation of crude initiation factors but absent in the crude IF-E, might be required for maxi- mal inhibition by the antibiotic.

M. Fresno. L. Carrasco, and D. Vazquer. 361

3

2

1

0 - . c - E ._ . LI) c

5 3 “ - c

4 z 5 2 * m E - % I

0

I

m

0

1.5

1 .o

0.5

0

D

10 20 30 40 0 10 20 30 40 50 Fraction number

Fig. 5 . Isohiion of 80-S-ribosonie[7sS]Met-tRNAl con7pks by Sephorose 6 B cohiniii chromaroRrupliy in the presence of’ crude inili- orion l f icturs. Conditions were as described in Methods. (A) Com- plete system; (B) minus crude initiation factors; (C) minus GTP; (D) minus poly(A,G,U); (E) 80-S ribosomes of Succi7uroinyces fereuisine insiead of 80-S rabbit reticulocyte ribosomes; (F) as (E) minus crude initiation factors

DISCUSSION

The number and specific action of eukaryotic initiation factors is not yet completely understood. Specific inhibitors might be very helpful in under- standing the mechanism of the initiation steps of protein synthesis. However the development of model systems is required for the elucidation of the action mechanism of an inhibitor to study partial reactions in which the inhibitor may be tested. We have de- veloped model systems to study some of the reactions taking place in the initiation of eukaryotic protein

synthesis (Fig. 8), in agreement with other workers [6,22]. For this purpose we have studied the binding of Met-tRNA, with a crude preparation of initiation factors or with a partially purified IF-E2 preparation. In a previous study [21] we observed that the factor involved in the binding of Met-tRNA, to ribosomes (IF-E2), absorbs to DEAE-cellulose and forms a ternary complex with Met-tRNA, and GTP, in agree- ment with other workers [ll - 141. Formation of this ternary complex is blocked by a number of inhibitors: adrenochrome, aurintricarboxylic acid, polydextran sulphate, pyrochatechol violet and showdomycin (Table 1). Aurintricarboxylic acid has no effect on the poly(A,G,U)-independent binding of Met-tRNA, to 40-S ribosomal subunits in the presence of a crude initiation factors preparation containing IF-E, . This is apparently an anomalous result if the formation of the ternary complex, which is blocked by aurintri- carboxylic acid, takes place prior to the binding of the initiator Met-tRNA, to the 4 0 3 ribosomal subunit. However in these experiments the 40-S ribosomal sub- units and the initiation factors were mixed and added to the reaction mixture before the addition of aurin- tricarboxylic acid and the reaction finally started by the addition of Met-tRNA,. Therefore it is likely that the factor IF-E, binds to the 40-S ribosomal subunit prior to the binding of Met-tRNA, and under these conditions remains protected against the action of the inhibitor. On the other hand aurintricarboxylic acid blocks the binding of Met-tRNA, to the 40-S ribo- somal subunit under conditions in which poly(A,G,U) was necessary, most probably because it prevents the binding of the messenger RNA to the small ribosomal subunit (for review see [7]); this is also the case of edeine Al . Chartreusin only partially affects this reaction, whereas polydextran sulphate was the most effective inhibitor.

The increased interaction of Met-tRNA, to the 4 0 3 subunit when the binding reaction to 80-S ribo- somes was studied in the presence of pactamycin might be an indirect effect due to the inhibition of the ‘join- ing reaction’ by the antibiotic, causing the accumula- tion of the smaller initiation complexes. However, pactaniin also increases the binding of Met-tRNA, with isolated 40-S subunits [5] . Our results indicate that most probably pactamycin fixes the initiator to the 40-S subunit making the inactive smaller initiator complex unable to go through the subsequent steps in initiation (Fig. 8).

Within the trichodermin group of antibiotics it has been postulated that some of them inhibit elongation (f. i. trichodermin) whereas some others inhibit in- itiation (f.i. verrucarin A) [23]. Fig. 6 shows that both trichodermin and verrucarin A enhance the binding of the initiator Met-tRNA, to the 80-S ribosome and both antibiotics inhibit peptide bond formation with [“Hlpuromycin. These results are in agreement with

362

1.5

1.4

1.3

1.2

1.1

I

1.4

E ).3 %

* r

01

c 1.2

9 32 4

).I

I

1.4

1.3

1.2

1.1

I

Fraction number

Fig. 6 . Analysis of [35S]Met-tRNA, . ribosome complex by sucrose gradient centrifugation in the presence of crude initiation factors. Conditions were as described in Methods. (A) Complete system; (B) minus crude initiation factors; (C) minus GTP; (D) plus 0.1 mM verrucarin A; (E) plus 0.1 mM trichodermin; (F) plus 0.1 mM trichothecin; (G) plus 0.1 mM sparsomycin; (H) plus 0.1 mM pactamycin; (I) ribosomes were preincubated with 10 mM N-ethylmaleimide for 10 min at 37 "C and the excess of inhibitor was neutralized with 2-mercaptoethanol, before the initiation of the reaction. Symbols as in Fig. 4

those found for the T-2 toxin [24] and indicate a similar mode of action for the different compounds of the trichodermin group of antibiotics. The only important difference between these compounds is that some of them such as verrucarin A appear to be unable to bind

to polysomal ribosomes and only interact with free ribosomes or 60-S ribosomal subunits then affecting only the first few peptide bonds (Table2). On the other hand trichodermin binds to both free or polysomal ribosomes [25], therefore inhibiting peptide bond

M. Fresno, L. Carrasco, and D. Vazquez 363

formation at any stage in translation. The enhanced binding of Met-tRNA, to the 80-S ribosomes is also observed with sparsomycin; however the site of action of trichodermin and sparsomycin on the 60-S ribo- somal subunit must be different because sparsomycin strongly increases the binding of C-A-C-C-A-Leu-Ac to the ribosome, whereas verrucarin A and trichoder- min do not have this effect (for review see [7]).

Table2. Effect of inhibitors on [35S]methiony1-puromycin formution The formation of [35S]methionyl-puromycin was carried out in three sequentional steps as described in Methods. When N-ethylmaleimide was tested, the ribosomes were preincubated for 10 rnin at 37 "C with the required concentration of the inhibitor and then the treated ribosomes were added to the reaction mixtures. ["S]- Methionyl-puromycin formation was 0.045 pmol with crude initiation factors and 0.085 pmol with crude IF-Ez

Inhibitor added Concen- [35S]Met-puromycin tration formed

+crude +crude initiation IF-E2 factors

mM % control

Verrucarin A 0.1 6 Trichodermin 0.1 18 Trichothecin 0.1 48 Pactamycin 0.1 0 Narciclasine 0.035 x 19 Sparsomycin 0.01 51 N-Ethylmaleimide 10 2 2-(4-Methyl-2,6 dinitro- ani1ino)-N-meth yl- propionamide 0.1 94 Pederine 0.1 C ycloheximide 0.1 94

-

2 15

67 8

55 7

-

15 112 -

IF-€,+ GTP+ Met - tRNAf I Adrenochrome Aurintricarboxilic acid Polydextran sulphate Pyrochatechol violet Showdornycin N-Ethylmaleirnide

IF-E,-GTP Met - tRNAf I

40 -S-subunit r '\ I Stabilization Met- tRNAf*40-S-subuni t !

Polydextran sulphate Edeine A, rnRNA Aurintricarboxilic acid IF-E,

Pactamycin ADP + P;

M e t - t R N A f ~ 4 0 - ~ - s u b u n i t mRNA Met- tRNAf *Pact -40-S-subun i t mRNA (Inactive and stable complex)

2 - ( 4 - Met hy I- 2,6- din i t roanilino) -

Sesqui t erpene antibiotics

NaF

Sparsomycin

60-S-subuni t

- N - methylpropionamide IF-E,

GDP +Pi

Purornycin

Methionyl -puromycin

Met- tRNAf -Antib-80-S-ribosorne-mRNA

Narciclasine Anisomycin Sesquiterpene antibiotics Sparsomycin

tRN A 80- S- ribosome mRN A

Fig. 8. A model scheme showing the steps of initiation of protein bio- synthesis in a mammalian system [6] und the mode of action of a number of inhibitors

Fraction number Fig. 7. Anulysis of [35S]Met-tRNA, . ribosome complexes in the presence of crude initiation.fiictor IF-E,. The formation of [35]Met-tRNA, . ri- bosome complex was carried out in two sequential steps. In the first one 160-p1 reaction mixtures containing 50 mM Tris-HCI buffer, pH 7.4, 100 mM KC1,8 mM 2-niercaptoethanol, 0.2 mM GTP, 0.32 pmol [35S]Met-tRNA, and 40 pg crude IF-E, were incubated at 37 "C for 10 min; 20 pl of a mixture containing 30 mM MgCI,, 0.8 A,,, unit of ribosomes and 6 pg poly(A,G,U) were then added, incubation continued for 10 rnin at 37 "C and the mixture was layered over 5-ml linear sucrose gradients (15-30%) as described in Methods. (A) Control; (B) as (A) but 20 pl of 10 mM puroinyciii were added to the mixture and incubation prolonged 10 min more. Symbols as in Fig.4

364 M. Fresno, L. Carrasco, and D. Vazquez: Inhibitors of Polypeptide Initiation in Eukaryotic Systems

It might be possible that the initiator Met-tRNAf binds initially to the A ribosomal site and in a further reaction concomitant with GTP hydrolysis the sub- strate is positioned in the P ribosomal site, through a translocation step. However the results presented in Table2 do not support this model since two trans- location inhibitors (pederine and cycloheximide) [7] have no effect on the formation of the first peptide bond. Therefore, it is likely that the initiator Met- tRNAf is directly positioned in the P ribosomal site.

Following the results presented in this paper, Fig. 8 shows schematically the steps into which the initiation may be divided in eukaryotic systems and the site of action of a number of translation inhibitors.

We are grateful to Miss Asuncion Martin for expert technical assistance. This work has been supported by Institutioncrl Grunts from Comisicin Adminixtradora clel De.scucwto Completnentarin (Instituto Nacional de Previsidn) and Direccion Genercrl de Sanidad (Spain).

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