J. Mol. Bid. (1976) 93,449-463
Ribosome Changes During Translation MARIANO BARBACID AND DAVID VAZQUEZ Institute de Biologia Celulur Vehzquez 144, Madrid 6, Spain (Received 12 August 1974, and in revised form 20 January
19Y5)
Studies on the quantitative binding of [sH]anisomycin are useful in determining conformational and/or structural changes on eukaryotic ribosomes. We have shown that yeast ribosomes have different structures depending on their functional states during the ribosome cycle as defined by their &inity for [3H]anisomycin. Free ribosomes, either in viwo run-off ribosomes (1 mu-sodium azide treatment or 8°C incubation of spheroplasts) or puromycin-dependent released ribosomes, have an affinity deilned by Kd = 3.3 to 3.6 pM. Ribosomes forming polysomes engaged in protein synthesis have at least two new different conformations (defined by KdmH= 0.81 PM and K,,, = 12 PM). These conformations have been ascribed to the pre and post-translocated steps of the elongation cycle in protein synthesis by blocking the polysomes with specific inhibitors of translation. Pre-translocated polysomes (polysomes blocked with cycloheximide) have an affinity of K, tax = 12 PM and post-translocated polysomes (polysomes blocked with doxycycline) have an afllnity of KdDC = 0.82 pM. These dissociation constants are identical to KdeL and K,,, obtained with control untreated polysomes, respectively. Moreover, a new ribosome conformation defined by KdDT = l-5 FM and KdFA = 13 pa6 was found, by blocking the polysomes with the elongation factor, EF-2, bound by using either diphtheria toxin or fusidio acid. We also present evidence of the previously reported heterogeneity of standard preparations of eukaryotic ribosomes (Barbacid & Vazquez, 1974a) being a direct consequence of the high-salt washing treatment of ribosomes.
1. Introduction Quantitative binding studies of the antibiotics [3H]anisomyoin and [3H]gougerotin to the peptidyl transferase centre of eukaryotic ribosomes (Barbacid Q Vazquez, 1974o,b) have shown that ribosomes prepared by the standard methods (“high-salt washed ribosomes”) from either yeast or human tonsils are structurally and functionally heterogeneous. This heterogeneity is not located on the antibiotic binding sites, not even on the larger ribosome subunit, which is the target for the above antibiotics, but is only observed when the entire ribosome is used. Only the ribosomes with a high a%nity for [3H]anisomycin are able to catalyse peptide bond formation studied by following the puromyoin reaction (Barbacid & Vazquez, 1974a). Taking advantage of the ability of [3H]anisomycin to distinguish different ribosomal structures, we have continued these binding studies in order to discover the conformational changes occurring on the ribosome during the ribosomel cycle and to explain the reasons for the observed heterogeneity of eukaryotic ribosomes washed 449
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in high-salt solutions. For these purposes we have developed a method of obtaining, on a preparative scale, polysome preparations without free ribosomes. We have specifically blocked these polysomes in different steps of the elongation cycle by using certain translation inhibitors. Finally we have studied the binding of [3H]anisomycin to the polysomes homogeneously blocked in different steps. Evidence for drastic conformational and/or structural changes of polysomes and ribosomes during protein synthesis is presented in this paper. In all cases such changes are defined by the parameters of the [3H]anisomycin interaction. Experiments with run-off ribosomes have shown that the high-salt washing treatment causes ribosomal heterogeneity.
2. Materials and Methods (a) Preparation
of yea& polysome8, ribo80me.3 and Buperndfznt fraction
(i) Yeast pO~y8O??W8 Polysomes from Sacc~romycea cerevisiae strain A224A were prepared from spheroplasts obtained following methods already described (Hutchison et al., 1969). All the experimental conditions described here lead to a batch of polysomes required for a single experiment of [3H]anisomycin binding. S. cerev&riue strain A224A was grown in 2 1 of yeaat/peptone/glucose medium (1% yeast extract, 2% peptone and 2% glucose) to an absorbance of 0.6 units at ,&so. The cells were harvested by centrifugation in a GS3 Sorvall rotor for 5 min at 10,000 g, washed with 1 vol. of sterile water, sedimented again by centrifugation and finally resuspended in O-1 of their volume of 1 M-sorbitol. Glusulase (END0 Laboratories) was added to a final concn of 0.6% and the suspension was incubated for 90 min at 30°C, with gentle shaking in order to obtain intact spheroplasts. These spheroplasts were then incubated for 3 h at 30°C in 1 vol. of YM-6 medium (Hartwell, 1967) (enriched with yeaat/peptone/glucose) and O-4 a4-MgSo, for the recovery of protein synthesis activity by the spheroplasts. During the last 10 min, cycloheximide (3 x 10e5 M) was present, except when otherwise indicated (e.g. untreated polysomes). The incubation of spheroplasts was suddenly stopped by mixing gently with O-5 vol. of 1 M-sorbitol (frozen at - 30°C) fragmented in small pieces. The spheroplasts were harvested by centrifugation in a GS3 Sorval rotor at 12,000 g for 30 min, and the pellet was then resuspended in l/60 vol. of lysis buffer (20 mna-TrismHCl buffer (pH 7*4), containing 30 mM-MgCl, and 100 mM-NaCl). Lysis was carried out with 2 sucoessive inoubations of 7 min at 0°C in the presence of sodium deoxycholate (0.20%) and Brij 35 (Sigma) (0.25%). The lysed extract was centrifuged in an SS34 Sorval rotor at 20,000 g for 15 min. Hereafter we shall refer to the supernatant of this centrifugation as the S-20. The S-20 was layered on a discontinuous sucrose gradient made with 10 ml of 60% sucrose and 6 ml of 20% sucrose in a “standard buffer” (20 mu-TriseHCl buffer (pH 7.4), containing 30 muMgCl, and 100 mu-KCl). After centrifugation in a 60Ti Spinco/Beckman rotor for 60 min at 48,000 revs/min (175,000 g) the tube was carefully placed into ice and the 7-ml sample from the bottom of the tube was taken out with a syringe provided with a long needle. When the supernatant fraction was required, 10 to 16 ml from the upper part of the tube were also liken separately. In all cases the remaining pert of the liquid phase was discarded, whereas the pellet was resuspended with the 7-ml sample initially taken from the bottom of the tube. This suspension was diluted with 3 vol. of the standard buffer, described above, and placed over 10 ml of 60% sucrose contained in the same standard buffer. The polysomes were spun down for 12 h at 70,000 g and the pellet was resuspended in the standard buffer and clarified by centrifugation at 20,000 g for 15 min. The resulting supernatant containing the polysomes was immediately used for a binding assay. For assays of amino acid incorporation activity, the polysomes were stored at -30°C and no loss of activity was detaoted either after defreezing and freezing three successive
times, or after two weeks of storage.
RIBOSOME All the media and Methods were sterilized that a monosome unit ml had an absorbance 40 mg of polysomes, of 0.36 units at A8s0.
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451
buffers described in sections (a), (b), (c) and (d) of Materials and before use. For the estimations of concentration it wss assumed had a molecular weight of 4 x lo6 and a solution of 1 mg of polysomes/ at 260 nm of 12 units. free of monoribosomes, were obtained per litre of cells in a culture was 1.80. The average ratio of &&A,,,
(ii) Yeast ribommes High-salt washed ribosomes were prepared according to methods already described for human tonsil ribosomes (Barbacid & Vazquez, 1974o). Run-off ribosomes were obtained from protein-synthesizing spheroplasts either treated with 1 ma6-sodium azide for 5 min or incubated at 8°C for 10 min. The method for preparing run-off ribosomes was basically the same as the method described for preparing polysomes. However, after the first highspeed centrifugation, free ribosomes remained in the interphase of the 20% and the SOY{, sucrose layers. Therefore, this part of the gradient was taken out to obtain run-off ribosomes instead of the part from the bottom of the tube as described in the polysome preparation. Finally, run-off ribosomes were spun down overnight (100,000 g) through a 20% sucrose layer in the standard buffer. The molecular weight and concentration/absorbance ratio were assumed to be similar, as in polysomes. The average values for the A 280/A280 ratio were 1.98 for high-salt washed ribosomes and 1.83 for run-off ribosomes. (iii) Supernatant
fraction
A crude extract, containing the elongation factors EF-1 and EF-2 and animoacyl-tRNA synthetases, was obtained from the upper part of the gradient resulting from the highspeed centrifugation of the S-20 extract (see section (a)(i) above) by precipitation with ammonium sulphate between 30 and 70% saturation. The precipitate was resuspended in a small amount of standard buffer, initially dialysed overnight against 1000 vol. of 20 mM-TriseHCl buffer (pH 7.4), containing 10 m&r-2-mercaptoethsnol, and finally dialysed for 4 h longer in a similar buffer, but containing only 5 mM-2-mercaptoethanol. It wss stored in small samples under liquid nitrogen. (b) In vitro
polysome treatment
A reaction mixture was prepared containing, in a final vol. of 25 ml: 50 n-nr-Tris*HCl buffer (pH 7*4), 125 mM-MgCl,, 80 mu-KCl, 1 mM-dithiothreitol (Sigma), 1 mM-ATP (Sigma), 0.08 mM-GTP (Sigma), 4 mM-creatine phosphate (Calbiochem) and 40 rg of creatine phosphokinase/ml (Calbiochem), 40 pg yeast tRNA/ml (General Biochemicals) and 0.02 mM of 20 amino acids (Calbiochem). The crude supernatant fraction was required for maximum incorporation activity (2.5 ml per 25ml incubation mixtures). Polysomes were added to a final concn of 25 mg/ml (750 A aao units per 25 ml). Finally the required protein synthesis inhibitor was present in t,he mixture as indicated in each case. The above reaction mixture was incubated at 30°C for 10 min, cooled to 0°C and layered on 10 ml of 60% sucrose in the standard buffer. Polysomes were spun down in a 60Ti Spinco/ Beckman rotor at 200,000 g for 4 h. They were resuspended in a small volume of standard buffer and clarified by centrifugation at low speed. The polysome concentration was determined and the preparation was immediately used for a binding assay. 75% of the initial polysomes were recovered. When polysomes were treated with puromycin, the incubation was carried out for 30 min and the released ribosomes were sedimented in the ultracentrifuge through a 20% sucrose layer.
(c) Polyaome activity (i) Amino acid incorporation
assay
The following reaction mixture was taken in a final vol. of 0.05 ml : 50 mna-Tris . HCl buffer (pH 7*4), containing 12.5 mM-Mg&, 80 m&r-KCl, 1 mM-dithiothreitol, 1 mnr-ATP, 0.08 mM-GTP, 4 mM-creatine phosphate, 40 pg creatine phosphokinase/ml, 40 rg yeast tRNA/ml and 0.02 mM of 19 amino acids (phenylalanine was omitted). [14C]Phenylalanine
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(New England Nuclear, 458 mCi/mmol) was added to a final concn of 0,015 mu, reducing its spec. act. 2.5 times. The crude supernatant fraction was added to detect a maximum of incorporation activity (usually 5 ~1). The reaction was initiated by adding 1.5 A,,, units of polysomes per tube. Incubation was at 30°C for the required time and was stopped by addition of l-5 ml cold trichloroacetic acid with 1% Casamino acids. Samples were heated for 15 min at 80 to 9O”C, cooled off, filtered through GFjC glass fibre Whatmail filters and radioactivity was determined. Va,lues in controls without polysomes wore subtracted in all cases. (ii) Peptidyl-puromyccin
a88ay
The following reaction mixture was taken in a final vol. of 0.05 ml: 50 mu-Tris .HC’l containing 125 mM-MgCl,, 80 mM-KCl, 0.5 rnivr-GTP, 5 ~1 of the crude supernatant fraction and 2.3 A,,, units of polysomes. The mixture was preincubated at 30°C for 7 min. 4 pM-[3H]pUrOmyCin (The Radiochemical Centre, Amersham, 3.7 Ci/mmol) was then added and the incubation was continued for 1 min more. The reaction was completed by addition of 1 ml of 10% cold trichloroacetic acid. Samples were filtered as previously described were used. Values (Pestka et al., 1972) except that GF/C glass fibre filters (Whatman) obtained in blank tubes, in which 10% trichloroacetic acid was added before [3H]puromycin, were subtracted in all cases. Inhibitors were added either at the beginning of the incubation (zero time) or simultaneously with puromycin (at 7 min) depending on the assay (see Results). (d) Sucrose gradient analysis Polysomes or ribosomes were analysed in 5.5-ml linear sucrose gradients of 15% to 300/, sucrose in standard buffer by centrifugation in an SW50.1 Spinco/Beckman rotor at 45,000 revs/min for 30 min and monitored in an ISCO model 183 gradient fractionator. Profiles of all the preparations described in the present work were plotted in the appropriate Figures.
(e) Quantitative
binding of [~H]ankomycin
Binding of [3H]anisomycin (260 mCi/mmol) (Barbacid & Vazquez, 1973) to ribosomcs was studied following the sedimentation method as previously described (Fernandezbuffer (pH 7*4), containing 30 m&I-MgCl, and Mufioz et al., 1971) in 20 mM-Tris.HCl 100 m&r-KC1 at 0°C. Data were plotted according to Scatchard (1949) and the dissociation constant K, is presented as a measure of the affinity of [3H]anisomycin for the ribosome. In all cases the ribosome is considered as a single unit even when it is bound t,o mRNA forming part of a polyeome.
3. Results (a) Ribosomd
heterogeneity of eukuryotic
ribosmes
A heterogeneity in standard preparations of washed eukaryotic ribosomes was found when studying the binding of [3H]anisomycin and [3H]gougerotin (Barbacid $ Vazquez, 1974a,b). The heterogeneity might be due either to differences in the functional states of the ribosomes or in their response to the high-salt treatment. In order to resolve this problem we have studied [3H]anisomycin binding to run-off ribosomes prepared from spheroplasts treated with 1 mM-sodium azide (Fig. l(a)) or incubated for ten minutes at 8°C (Fig. l(b)). Under these experimental conditions the initiation process of translation is inhibited in vivo. Therefore, polysomes are not formed and all ribosomes are free of messenger RNA. As shown in Figure l(a) and (b) these ribosomes are homogeneous for [3H]anisomycin binding. Very similar results were obtained with both types of run-off ribosome preparations. At saturation, we may take it that one molecule of
RIBOSOME
CHANGES
DURING
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453
[3H]anisomycin is bound per ribosome. The aflinity value was Ktboff = 3.5 x 10m6M for ribosomes prepared from sodium azide-treated spheroplasts and Kz-Off = 3.3 x 10m6 M for ribosomes prepared from spheroplasts incubated at 8°C. The parameters indicated above for [3H]anisomycin binding differ from those previously found with standard washed ribosomes (Barbacid & Vazquez, 1974a). Since a different strain of yeast was used before, we have now studied [3H]anisomycin
FIN. 1. Scatchard plots of the data for [sH]anisomycin binding to free ribosomes obtained from : (a) 1 mr+r-sodium azide-treated yeast spheroplasts; (b) 8°C incubated yeast spheroplasts; (c) yeast cells, following standard methods (“high-salt washed ribosomes”) (Barbacid & Vazquez, 1974a); and (d) 1 mna-sodium azide-treated yeast spheroplasts, followed by a high-salt washing treatment similar to that in (c). In all cases the binding assays were carried out by the ultracentrifugation method at 0°C (140,000 g, 2 h) under the following ionic oonditions: 60 man-Tris.HCl buffer (pH 7.4), containing 30 mM-MgCl, and 100 mM-KCl. Ribosome concentrations were 3 x 10-s M for tubes with [sH]anisomycin comma ranging from 2 x 10-r 116to 2 x 10-s M and 6 x 10-s M for tubes with [3H]vols were 80 and 60 4, anisomyoin concns ranging from 2 x 10-s M to 2.6 x 10e5 M. Incubation respectively, and samples (20 and 10 4, respectively) were taken before and after sedimentation of the ribosomes. The profiles indicate the sucrose gradient analysis (A,,,) of each ribosome or polysome preparation.
binding to standard washed ribosomes from the S. cerevisiae strain A224A, which was used throughout this work. For this purpose yeast was grown in a 20-litre fermentor, growth was stopped by addition of ice at the logarithmic phase and cells harvested by continuous centrifugation. Washed ribosomes were obtained (see Materials and Methods) and [3H]anisomycin binding was studied (Fig. l(c)). Approximately 30% of the ribosomes bind the antibiotic with a higher affinity defined by KffFW = 5.2 x 10e7 M, whereas the remaining 70% interact with a lower affinity (K@”,- = 5x 10d6 M). These results are practically identical to those obtained 30
454
M. BARBACID
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VAZQUEZ
previously with similar ribosome preparations from X. cerewisiue strain PM-l, if the changes introduced in the ionic conditions are taken into account (see Fig. 1 in Barbacid t Vazquez, 1974a). However, the differences between the results presented in either Figure l(a) or (b) and Figure l(c) might be due to the high-salt treatment or the cell-disrupting procedure. Therefore, a ribosome preparation, identical to that used in the experiment shown in Figure l(b), was washed with 0.5 IN-NH&I and 0.1 M-MgCl,. The results obtained when [3H]anisomycin binding to these ribosomes was studied, clearly she\\ that two different types of ribosomes were obtained after high-salt treatment of run-off ribosomes (Fig. l(d)). However, in this case the percentage of each type of ribosome and the value of the dissociation constant of the higher affinity ribosomes differ considerably from those of Figure l(c) (K,,, = 14~ 10e6 and y1 = O-37: K,,, = 5.6 x lOa and v2 = 0.48. These results might indicate that the parameters of the heterogeneous binding are somehow dependent on the treatment of the cells prior to the isolation of the ribosomes. (b) Preparation and activity of polysomes It is generally accepted that ribosomes undergo conformational changes during the steps of the ribosomal cycle (Haselkorn $ Rothman-Denes, 1973, review). The ability of [3H]anisomycin to distinguish between the structure or conformation of run-off and high-salt treated ribosomes, encouraged us to study [3H]anisomycin binding to polysomes at different well-defined steps in order to detect any possible ribosomal changes. This required the preparation of polysomes without free ribosomes, which was achieved by a cycloheximide (3x 10m5 M) pre-treatment of the spheroplasts followed by centrifugation of the polysomes through a cushion of 60% sucrose. Our polysomes synthesized polypeptide chains linearly for at least 15 minutes and an average of 30 to 35 amino acids per ribosome was incorporated in 45-minute incubations (results not shown). (c) [3H]anisomycin binding to untwxzted polysomes Polysomes from control untreated spheroplasts show at least two different types of structure according to their affinity for [3H]anisomycin (Fig. 2). One third of the total polysomes interacting with the antibiotic show a high-affinity binding (K,,, = 8.1 x 10e7 M), whereas the other two-thirds of the polysomes have a lower affinity W d.L -- I.2 x 10m5 M). In this assay the [3H]anisomycin interaction with free ribosomes (approx. 15% of the total preparation) cannot be detected because the value of their dissociation constant is 3.5 x 10e6 M. Therefore, the percentage of polysomes with low affinity (K,,,) calculated from the Scatchard plot (Fig. 2) is probably higher than the real value. As shown before with other ribosome preparations, one molecule of antibiotic is bound per ribosome. (d) Peptidyl-puromycin fotiion This polysome heterogeneity might well reveal two different ribosomal stages belonging to two different steps of the elongation process in protein biosynthesis. Therefore, we developed a system for translocation and peptide bond formation based on that described by Pestka et al. (1972) with some important modifications, especially in the ionic conditions.
RIBOSOME
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466
In agreement with Pestka et al. (1972) we observed that a maximum of reactivity is found between 0.8 and 1-O ~-Kc1 almost independently of magnesium concentration. However, the crude supernatant fraction is able to stimulate the reaction as a consequence of a translocation process only at KC1 concentrations lower than 300 mM
0.6
0.8
I I.0
FICA 2. Soatohard plot of the data for [3H]anisomyain binding to untreated yeast polysomes. Data were obtainedfrom an ultrecentrifugation assay (14O,OOOg, 2 h) carried out at 0°C. 3 x 10Tg~polysomes were added for an [3H]anisomyain concn range of 0.26 to 2 x 10eB M, and 6 x 1OYp M were present for an [sH]anisomycin conan range of 2 x 1Om8 to 2.6 x 10m5 M. Ionic conditions and incubation volumes as in legend to Fig. 1. The profile shows the polysome content of t,he preparation by mesm of a suorose gradient analysis.
(unpublished results). Therefore, we used the same ionic conditions as described for amino acid incorporation by the polysomes. In all cases [3H]puromycin (4 x 1Oe6 M) was added after translocation. The effect of an inhibitor present at the beginning of the reaction reflects its effect on translocation and/or peptide bond formation. If the inhibitor was added just after the translocation preincubation, its effect on peptide bond formation only could be measured. The difference between both systems will give the effect of the inhibitor on tranalocation. Results are shown in Table 1. Diphtheria toxin, fusidic acid and cycloheximide appear to be inhibitors of translocation whereas sparsomycin completely inhibits peptide bond formation. Doxycycline affects neither reaction. These results are in agreement with the generally accepted mechanism of action of these inhibitors except in the case of fusidic acid (see Discussion) (Gale et al., 1972 ; Vazquez, 1974, reviews).
(0.012 mM) (O-01 man) (1 m4 (1 mx) (0.2 mma) (0.02 mM) 1.64 1.43 1.43 1.61 o-01
1.49
0 4 10 0 99
0
0.68 0.68 0.69 1.46 0.02
1.49 0.62
Pep?y:PM formation (pmol)
Peptidyl-transferase plus translocation
0.86 0.76 0435 0.08 -0.01
0 0.87
(pm4
Peptidyl-tRNA not translocated
99 86 75 7 0
0 100
o/0 Inhibition
Translocation
The peptidyl-transferase assay (1st column) was carried out after a prior translocation of the pept’idyl-tRNA from the A-site to the P-site by preincubation of the polysomes for 7 min at 30°C in the presence of a crude supernatant fraction and GTP. In the peptidyl-transferase plus translocation assay (2nd part of the Table), the inhibitors were present before the translocation. The 3rd part of the Table (peptidyl-tRNA blocked in the A-site) was obtained from the differences between the first and second parts, and hence the inhibition of the translocation reaction was calculated. The experimental conditions were as follows: 50 m&r-Tris.HCl buffer (pH 7+4), 12.5 mM-MgCl,, 80 mar-KCl, 0.5 m&r-GTP, 5 ~1 of a crude supernatant fraction, 2.3 Aleo units of polysomes and 4 PM-[aH]puromycin (spec. act. 3.7 Ci/mmol) in a 0.06 ml final vol. The translocation reaction was initiated with polysomes. The subsequent puromycin (PM) reaction was started by adding the labelled puromycin and incubating for a further minute also at 30°C. Reaction was stopped by the addition of 1 ml of cold 10% trichloroacetic acid and filtered through GF/C Whatman glass fibre filters as previously described (Pestka et al., 1972). The polysome preparation contained less than 15% of free ribosomes.
Complete - Supernatant + Diphtheria toxin plus NAD + Fusidio acid + Cycloheximide + Doxycycline + Sparsomycin
1
formation by translocated yeast polysomes
Peptidyl-transferase assay after translocation Peptidyl-PM formation o/0 Inhibition (pm4
Peptidyl-puromycin
TABLE
RIBOSOME
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457
(e) [3H]oni.sonaycin binding to in vitro treated polysomes (i) Cyclohexinaide pooolyaonaes Cycloheximide stops protein synthesis, most probably aa a consequence of a single interaction with the ribosome. Hence, it was expected that all the cycloheximidetreated polysomes had been blocked in the same elongation step in protein synthesis. Our results obtained by means of the puromycin reaction suggest that cycloheximide blocks polysomes with peptidyl-tRNA bound to their A-site. This is in agreement with previous experiments by other workers (Baliga et al., 1969,197O; Obrig et al., 1971). The results obtained from studying [3H]anisomycin binding to cycloheximide(10e3 M) treated polysomes are shown in Figure 3. All the polysomes with binding activity are homogeneous, showing an atEnity for the antibiotic (KdCHX = 1.2 x lo- 5 M) identical to the low-affinity binding of untreated polysomes (K,,, = 1.2 x 10v7 M). The saturation value (J) of cycloheximide-treated polysomes was lower than that of untreated polysomes. However, similar low values of 9 were obtained with polysomes treated in vitro with other antibiotics (see results presented below) suggesting that parts of the polysomes were damaged.
Fra. 3. So&chard plot for [sH]anisomyoin binding to yeast oyoloheximide-treated polysomes. The in vitro treatment was oarried out by inoubating the polysomes for 10 min at 30°C in the presenoe of 1 mna-oyoloheximide as described in Matarials and Methods. Polysomes isolated by sedimentation through a 60% suoroae layer were immediately taken for the ultraoentrifugation binding assay. Polysome ooncentrations were 3 and 6 x 10-O I for [sH&nisomyoin oonon ranges of 1 to 3 x 10Te M and 3 x 10-s M to 3 x 10e6ar, respeotively. Otherwise the experimental conditions were as indicated in the legend to Fig. 1.
(ii) Doxycycline polysmaes In order to study the binding of anisomycin to polysomes with peptidyl-tRNA bound to the P-site, polysomes were treated in vitro with O-2 rnlcl-doxycycline for ten minutes at 30°C to inhibit aminoacyl-tRNA binding to the A-site, and therefore block peptidyl-tRNA in the P-site (Gale et al., 1972; Vazquez, 1974, reviews). As Figure 4 shows, these ribosomes were heterogeneous for [3H]anisomycin binding. However, up to 60% of the active polysome fraction bound the antibiotic with the higher affinity (Kt,: = O-82x 10ee M) which differs from the low-affinity binding of polysomes with the peptidyl-tRNA bound to the A-site.
488
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Y
FIG. 4. Scatchard plot of d&a for [3H]anisomycin binding to yeast doxycycline-treated polysomes. The in vitro inoubation was oarried out at 30°C for 10 min in the presenoe of 0.2 mMdoxycychne. After sedimentation, polysomes were immediately taken for the following binding assay: polysome ooncentrations were 2.5 x 10eB M (for tubes with [3H]anisomycin concns ranging from 0.2 to 1 x 1O-e M), 4~ 10m3 M ([3H] anisomycin conctns from 1 x 10eB to 1 x 1Ou5 Y) and 6 x 10-s M ([3H]anisomyoin concns from 1 to 4 x 10-s M). For other experimental aonditions see legend to Fig. 1.
(iii) Diphtheria; toxilt and fmidic acid polysomes It was also of interest to study the interaction of [3H]anisomycin with polysomes carrying stably bound elongation factor EF-2. This was possible by treating polysomes in the presence of GTP and EF-2 with either diphtheria toxin + NAD or fusidic acid, which promote the formation of either ADP-ribosyl-EF-2 *GDP-ribosome or EF-2 efusidic acid* GDP-ribosome, respectively. In the first complex peptidyl-tRNA is located in the A-site. Similarly, the substrate is not reactive with puromycin in the fusidic acid-stabilized complex. Polysomes were incubated at 30°C for ten minutes in the presence of either 12 pMdiphtheria toxin plus 10e5 M-NAD or 1 rnllr-fusidic acid, and subsequently isolated by sedimentation through a 60% sucrose layer, which contained 1 mm-fusidic acid when present in the incubation mixture. With both complexes, homogeneity of [3H]anisomycin binding was observed (Fig. 5) suggesting that all the polysomeswere blocked at the same step. As with other in vitro treated polysomes, the saturation values were lower than 1, most probably due to partial inactivation during the preparation procedure. The most important feature of these experiments is the almost identical value of the dissociation constant in both cases. From Figure 5(a) an ailinity
RIBOSOME
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459
Fm. 6. Scatchard plots of the data for [sH]anisomycin binding to polysomes pretreated with either: (a) diphtheria toxin; or (b) fusidic acid. The in vitro treatments were carried out as described in Materials and Methods in the presence of 12 PM-diphtheria toxin plus 1 x 10m5 M-NAD or 1 mM-fusidia acid. All the experimental conditions concerning the binding assay were as indiaated in the legend to Fig. 1. The polysome profiles of the preparations used for [3H]anisomycin binding are shown as in all the Figures.
of KaDT = 1.8 >: 10 - 6 M can be deduced for the interaction between [“HJanisomycin and diphtheria toxin-treated polysomes. A dissociation constant of KaFA = 1.5 x 10s6 M was found (Fig. 5(b)) in the caseof fusidic acid treatment. (iv) Puromycin-released ribosomes When polysomes were incubated for 30 minutes at 30°C in the presence of 1 mMpuromycin under conditions in which they are actively incorporating amino acids, a breakdown of polysomes into monosomes occurred. We measured the ability of those puromycin-released monosomes to interact with [3H]anisomycin in order to compare them with run-off or high-salt washed ribosomes. The Scatchard plot of Figure 6 clearly shows a homogeneity with a saturation value of 0.6. The resulting dissociation constant KdPM = 3.6 x 10e6 M is practically identical with that of in vivo run-off ribosomes (either sodium azide- or cold-treated spheroplasts, Fig. 1).
4. Discussion Our results show that conformational changes of the ribosome during the process of translation can be detected by quantitative studies of [3H]anisomycin binding. Furthermore, we have shown that high-salt washing of the ribosomes, by the standard methods used to separate the different protein factors required for the process of translation, also causes some conformational or structural changes that lead to heterogeneous ribosome populations. Previously (Barbacid BEVazquez, 1974a) we have demonstrated that these changes mainly affect the 40 S eukaryotic ribosome subunit.
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D. VAZQUEZ
FIR 6. Soatchard plot for [3H]anisomycin binding to ribosomes released from polysomes by in vitro puromyain treatment. The in vitro treatment only differed from those aarried out with other inhibitors in thet the incubation time was 30 min. Puromycin-released ribosomes were isolrtted by sedimentation in the ultraaentrifuge through 8 20% sucrose layer instead of a 60% layer as in the case of the in vitro treated polysomes. The ultraoentrifugation binding assay was aarried out identically to those described in the legend to Fig. 1.
In a simplified model of protein synthesis, ribosomes can be found in two different stages: the free ribosomes (between termination and initiation) and the polysomes (engaged in translation). The present study of [3H]anisomycin binding to these two types of ribosomes leads to the following conclusions: (1) free ribosomes (“run-off ribosomes”) are homogeneous with regard to anisomycin binding, and (2) they differ from high-salt washed ribosomes (Fig. l), and (3) from polysomes (Fig. 2), which are heterogeneous. The heterogeneity of polysomes with regard to anisomycin binding is due to at least two distinct conformational states of ribosomes during the elongation cycle. From the results obtained with [3H]anisomycin binding to cycloheximide-treated polysomes, it is clear that: (1) cycloheximide blocks the polysomes in a defined state (Fig. 3); and (2) this state, which corresponds to the phase in which the peptidyltRNA is bound to the A-site (Table I), is one of those found with untreated polysomes (low-affinity binding, K,,, = KdCHX= 12 PM). Doxycycline-treated polysomes do not show total homogeneity. This might be due to the small inhibitory effect of the tetracycline group of antibiotics on aminoacyltRNA binding in eukaryotic cell-free systems. It is suggested that the high-affinity binding defined by K,DC 1 - O-82x 10 - ’ M (up to 60% of polysomes) corresponds to the state in which peptidyl-tRNA is bound to the ribosomal P-site (Fig. 7). As this value is practically identical to K,., = 041 x 1Oms M, we can ascribe the second structure found with untreated polysomes (the high-affinity binding) to the step blocked by doxycycline. As untreated polysomes were obtained from spheroplasts rapidly cooled at O”C, it seems as if aminoacyl-tRNA binding and translocation are the most cold-sensitive steps in the elongation process of protein synthesis in yeast. It should be taken into
RIBOSOME
CHANGES
DURING
TRANSLATION
461
Diphtheria toxin or Fusidic acid
Cycloheximide
Y Termination Elongation cycle
K,,L=Kd CHx= 12/&M
z
FIG. 7. Scheme of the ribosome NaAz, sodium azide.
32
U
K,,“=K:‘=
O+M
A Doxycycline
oyole studied
by [3H]anisomyoin
binding.
PM, puromyoin;
account that variations in the ionic conditions during the polysome isolation procedure could modify the proportions of pre and post-translocated ribosomes found in our untreated polysome preparations (Fig. 2). Fusidic acid is widely accepted as an inhibitor of aminoacyl-tRNA binding in intact bacteria and in several cell-free systems (Gale et al., 1972; Vazquez, 1974). Theee results have been explained by the blocking of the ribosomal A-site by the EF-G (or EF-2) *GDP*ribosome complex which is stabilized by fusidic acid. The previously reported inhibitory etIeot of fusidic acid on translocation in model prokaryotic and eukaryotic cell-free systems has been explained on the basis of a trapping of all the available EF-G or EF-2 by the free ribosomes in the reaction mixture (Vazquez, 1974). However, the inhibition on trenslocation by fusidic acid observed in our pure polysomal system (Table 1) cannot be due to depletion of EF-2 through such a trapping effect because: (1) the fusidic acid-treated polysomes themselves seemed to interact with EF-2, as evident from their changed affinity for [3H]anisomycin upon the addition of the crude supernatant fraction in the presence of fusidic acid (Fig. 5(b), see below) ; and (2) increasing amounts of the supernatant fraction (15 times) did not reverse the inhibitory effect of fusidic acid (results not shown), contrary to the results which we obtained previously in a model ribosomal system (Vazquez, 1974). Our results from both [3H]anisomycin binding (Fig. 5) and peptidyl-transferase studies (Table 1) strongly suggest that fusidic acid and diphtheria toxin block polysomes in a very similar, if not identical, defined conformation. Both reagents, like cycloheximide, prevent functional trsnslocation as measured by puromycin reactivity of peptidyl-tRNA (Table 1). In the case of diphtheria toxin, peptidyl-tRNA remains in the A-site; in the case of fusidic acid, the position of peptidyl-tRNA has not yet been defined. Unlike cycloheximide, however, diphtheria toxin and fusidic acid confer on polysomes a high affinity to anisomycin. This is presumably due to the stable (EF-2eribosome) complex formation known to result with diphtheria toxin (Bermek, 1972) and fusidic acid (Gale et al., 1972; Vazquez, 1974). From the almost identical dissociation constants of anisomycin for diphtheria toxin- and fusicid acid-treated
462
M. BARBACID
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D. VAZQUEZ
polysomes (KdDT = 18x 10m6 M and K dFA = 1*5X 10e6 M), it appears that the ADP-ribosylation of EF-2 by diphtheria toxin does not modify its interaction with polysomes. Considering the above results, it would be of interest to study [3H]anisomycin binding to polysomes with peptidyl-tRNA bound to the P-site and aminoacyl-tRNA bound to the A-site. This polysomal preparation might be obtained “a priori” by freezing the polysomes with inhibitors of peptide bond formation. However, we have observed that yeast polysomes blocked with a peptide bond formation inhibitor like blasticidin S (Vazquez, 1974) behave similarly as control polysomes for [3H]anisomycin binding (results not shown). This result might be expected since peptidyl-transferase inhibitors reversibly block this ribosome centre. Therefore, when the inhibitor is not bound, peptidyl-transferase immediately catalyses peptide bond formation and polysomes remain heterogeneous in the same way as untreated polysomes. Sparsomycin, a very effective inhibitor of peptide bond formation (Table l), could not be used, since when it is bound to polysomes it strongly inhibits [3H]anisomycin binding. Indeed, with sparsomycin-treated polysomes, only 8% of the total polysome preparation bound [3H]anisomycin. The dissociation constant was found to be around 3 PM. This interaction must undoubtedly correspond to the small fraction of free ribosomes. The rest did not bind the labelled antibiotic, at least with a dissociation constant lower than 100 pM. Known specific inhibitors of eukaryotic peptide bond formation cannot be used because of their inhibitory effect on [3H]anisomycin binding (Barbacid & Vazquez, 1974u). The results discussed above can be summarized as indicated in Figure 7. The dissociation constants for [3H]anisomycin binding corresponding to the different conformational and/or structural states of the ribosomes during the process of translation are as follows: (1) free ribosomes (Kf-“’ = 3.5 PM); (2) post-translocated ribosomes (K$ or K,,, = 0.8 PM); (3) pre-translocated ribosomes (KdCHX or K,,, - 12 PM); and (4) polysomes with EF-2 bound (KdDT or .KdFA = 1.8 to 1.5 pM). It should be pointed out that the highest affinity for [3H]anisomycin binding was observed with post-translocated ribosomes (polysomes with peptidyl-tRNA bound to the P-site) which is precisely the step in which the antibiotic should be bound to the ribosome in order to exert its effect by blocking peptide bond formation. We thank Dr J. Modolell for his interest and for criticism of the manuscript. We am also grateful to Dr A. Jimenez and L. Sanchez for their advice in the polysome preparation procedure and to Miss A. Martin for expert technical assistance. This work has been supported by grants from Fondo National para el Desarrollo de la Investigation Cientifica, U.S. National Institutes of Health (A108598) and Lilly Indiana of Spain. One of us (M. B.) was the holder of a fellowship of Plan de Formation de Personal Investigador.
REFERENCES Baliga, B. S., Pronczuk, A. W. & Munro, H. N. (1969). J. Bid. Chem. 244, 4480-4489. Baliga, B. S., Cohen, S. A. & Munro, H. N. (1970). FEBS Letters, 8, 249-252. Barbacid, M. & Vazquez, D. (1973). Anal. Biochem. 56, 18-25. Barbacid, M. & Vazquez, D. (1974a). J. Mol. Biol. 84, 603-623. Barbacid, M. & Vazquez, D. (19743). Eur. J. Biochem. 44, 445-453. Bermek, E. (1972). FEBS Letters, 23, 95-99. Fernandez-Mtioz, R., Monro, R. E., Torres-Pinedo, R. & Vazquez, D. (1971). I&T. Biochern. 23. 185-193.
J.
RIBOSOME Gale, E. P., Cundliffe,
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463
E., Reynolds, P. E., Richmond, M. H. Jz Waring, M. J. (1972). Ln of Antibiotic Action, pp. 278-379, John Wiley & Sons, London. Hartwell, L. H. (1967). J. Bactertil. 93, 1662-1670. Haselkorn, R. & Rothman-Denes, L. (1973). Alznu. Rev. Biochem. 42, 397-438. Hutchison, H. T., Hartwell, L. H. & McLaughlin, C. S. (1969). J. Bacterial. 99, 807-814. Obrig, T. G., Gulp, W. J., McKeehan, W. 1,. & Hardesty, B. (1971). J. Bid. Chew.. 246, 17P181. Pestka, S., Goorha, R., Rosenfeld, H., Neurath, C. 8: Hintikkn, H. (1972). -7. Bfiol. C!I/cvv. 247, 4258-4263. Scatchard, G. (1949). Ann. N.Y. Acad. Sci. 51, 660-672. Vazquez, I). (1974). PEBS Letter,?, 40, suppl. S63-S84. The Molecular
Basis
Ribosome Changes During Translation MARIANO BARBACID AND DAVID VAZQUEZ Institute de Biologia Celulur Vehzquez 144, Madrid 6, Spain (Received 12 August 1974, and in revised form 20 January
19Y5)
Studies on the quantitative binding of [sH]anisomycin are useful in determining conformational and/or structural changes on eukaryotic ribosomes. We have shown that yeast ribosomes have different structures depending on their functional states during the ribosome cycle as defined by their &inity for [3H]anisomycin. Free ribosomes, either in viwo run-off ribosomes (1 mu-sodium azide treatment or 8°C incubation of spheroplasts) or puromycin-dependent released ribosomes, have an affinity deilned by Kd = 3.3 to 3.6 pM. Ribosomes forming polysomes engaged in protein synthesis have at least two new different conformations (defined by KdmH= 0.81 PM and K,,, = 12 PM). These conformations have been ascribed to the pre and post-translocated steps of the elongation cycle in protein synthesis by blocking the polysomes with specific inhibitors of translation. Pre-translocated polysomes (polysomes blocked with cycloheximide) have an affinity of K, tax = 12 PM and post-translocated polysomes (polysomes blocked with doxycycline) have an afllnity of KdDC = 0.82 pM. These dissociation constants are identical to KdeL and K,,, obtained with control untreated polysomes, respectively. Moreover, a new ribosome conformation defined by KdDT = l-5 FM and KdFA = 13 pa6 was found, by blocking the polysomes with the elongation factor, EF-2, bound by using either diphtheria toxin or fusidio acid. We also present evidence of the previously reported heterogeneity of standard preparations of eukaryotic ribosomes (Barbacid & Vazquez, 1974a) being a direct consequence of the high-salt washing treatment of ribosomes.
1. Introduction Quantitative binding studies of the antibiotics [3H]anisomyoin and [3H]gougerotin to the peptidyl transferase centre of eukaryotic ribosomes (Barbacid Q Vazquez, 1974o,b) have shown that ribosomes prepared by the standard methods (“high-salt washed ribosomes”) from either yeast or human tonsils are structurally and functionally heterogeneous. This heterogeneity is not located on the antibiotic binding sites, not even on the larger ribosome subunit, which is the target for the above antibiotics, but is only observed when the entire ribosome is used. Only the ribosomes with a high a%nity for [3H]anisomycin are able to catalyse peptide bond formation studied by following the puromyoin reaction (Barbacid & Vazquez, 1974a). Taking advantage of the ability of [3H]anisomycin to distinguish different ribosomal structures, we have continued these binding studies in order to discover the conformational changes occurring on the ribosome during the ribosomel cycle and to explain the reasons for the observed heterogeneity of eukaryotic ribosomes washed 449
450
M. BARBACID
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D. VAZQUEZ
in high-salt solutions. For these purposes we have developed a method of obtaining, on a preparative scale, polysome preparations without free ribosomes. We have specifically blocked these polysomes in different steps of the elongation cycle by using certain translation inhibitors. Finally we have studied the binding of [3H]anisomycin to the polysomes homogeneously blocked in different steps. Evidence for drastic conformational and/or structural changes of polysomes and ribosomes during protein synthesis is presented in this paper. In all cases such changes are defined by the parameters of the [3H]anisomycin interaction. Experiments with run-off ribosomes have shown that the high-salt washing treatment causes ribosomal heterogeneity.
2. Materials and Methods (a) Preparation
of yea& polysome8, ribo80me.3 and Buperndfznt fraction
(i) Yeast pO~y8O??W8 Polysomes from Sacc~romycea cerevisiae strain A224A were prepared from spheroplasts obtained following methods already described (Hutchison et al., 1969). All the experimental conditions described here lead to a batch of polysomes required for a single experiment of [3H]anisomycin binding. S. cerev&riue strain A224A was grown in 2 1 of yeaat/peptone/glucose medium (1% yeast extract, 2% peptone and 2% glucose) to an absorbance of 0.6 units at ,&so. The cells were harvested by centrifugation in a GS3 Sorvall rotor for 5 min at 10,000 g, washed with 1 vol. of sterile water, sedimented again by centrifugation and finally resuspended in O-1 of their volume of 1 M-sorbitol. Glusulase (END0 Laboratories) was added to a final concn of 0.6% and the suspension was incubated for 90 min at 30°C, with gentle shaking in order to obtain intact spheroplasts. These spheroplasts were then incubated for 3 h at 30°C in 1 vol. of YM-6 medium (Hartwell, 1967) (enriched with yeaat/peptone/glucose) and O-4 a4-MgSo, for the recovery of protein synthesis activity by the spheroplasts. During the last 10 min, cycloheximide (3 x 10e5 M) was present, except when otherwise indicated (e.g. untreated polysomes). The incubation of spheroplasts was suddenly stopped by mixing gently with O-5 vol. of 1 M-sorbitol (frozen at - 30°C) fragmented in small pieces. The spheroplasts were harvested by centrifugation in a GS3 Sorval rotor at 12,000 g for 30 min, and the pellet was then resuspended in l/60 vol. of lysis buffer (20 mna-TrismHCl buffer (pH 7*4), containing 30 mM-MgCl, and 100 mM-NaCl). Lysis was carried out with 2 sucoessive inoubations of 7 min at 0°C in the presence of sodium deoxycholate (0.20%) and Brij 35 (Sigma) (0.25%). The lysed extract was centrifuged in an SS34 Sorval rotor at 20,000 g for 15 min. Hereafter we shall refer to the supernatant of this centrifugation as the S-20. The S-20 was layered on a discontinuous sucrose gradient made with 10 ml of 60% sucrose and 6 ml of 20% sucrose in a “standard buffer” (20 mu-TriseHCl buffer (pH 7.4), containing 30 muMgCl, and 100 mu-KCl). After centrifugation in a 60Ti Spinco/Beckman rotor for 60 min at 48,000 revs/min (175,000 g) the tube was carefully placed into ice and the 7-ml sample from the bottom of the tube was taken out with a syringe provided with a long needle. When the supernatant fraction was required, 10 to 16 ml from the upper part of the tube were also liken separately. In all cases the remaining pert of the liquid phase was discarded, whereas the pellet was resuspended with the 7-ml sample initially taken from the bottom of the tube. This suspension was diluted with 3 vol. of the standard buffer, described above, and placed over 10 ml of 60% sucrose contained in the same standard buffer. The polysomes were spun down for 12 h at 70,000 g and the pellet was resuspended in the standard buffer and clarified by centrifugation at 20,000 g for 15 min. The resulting supernatant containing the polysomes was immediately used for a binding assay. For assays of amino acid incorporation activity, the polysomes were stored at -30°C and no loss of activity was detaoted either after defreezing and freezing three successive
times, or after two weeks of storage.
RIBOSOME All the media and Methods were sterilized that a monosome unit ml had an absorbance 40 mg of polysomes, of 0.36 units at A8s0.
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451
buffers described in sections (a), (b), (c) and (d) of Materials and before use. For the estimations of concentration it wss assumed had a molecular weight of 4 x lo6 and a solution of 1 mg of polysomes/ at 260 nm of 12 units. free of monoribosomes, were obtained per litre of cells in a culture was 1.80. The average ratio of &&A,,,
(ii) Yeast ribommes High-salt washed ribosomes were prepared according to methods already described for human tonsil ribosomes (Barbacid & Vazquez, 1974o). Run-off ribosomes were obtained from protein-synthesizing spheroplasts either treated with 1 ma6-sodium azide for 5 min or incubated at 8°C for 10 min. The method for preparing run-off ribosomes was basically the same as the method described for preparing polysomes. However, after the first highspeed centrifugation, free ribosomes remained in the interphase of the 20% and the SOY{, sucrose layers. Therefore, this part of the gradient was taken out to obtain run-off ribosomes instead of the part from the bottom of the tube as described in the polysome preparation. Finally, run-off ribosomes were spun down overnight (100,000 g) through a 20% sucrose layer in the standard buffer. The molecular weight and concentration/absorbance ratio were assumed to be similar, as in polysomes. The average values for the A 280/A280 ratio were 1.98 for high-salt washed ribosomes and 1.83 for run-off ribosomes. (iii) Supernatant
fraction
A crude extract, containing the elongation factors EF-1 and EF-2 and animoacyl-tRNA synthetases, was obtained from the upper part of the gradient resulting from the highspeed centrifugation of the S-20 extract (see section (a)(i) above) by precipitation with ammonium sulphate between 30 and 70% saturation. The precipitate was resuspended in a small amount of standard buffer, initially dialysed overnight against 1000 vol. of 20 mM-TriseHCl buffer (pH 7.4), containing 10 m&r-2-mercaptoethsnol, and finally dialysed for 4 h longer in a similar buffer, but containing only 5 mM-2-mercaptoethanol. It wss stored in small samples under liquid nitrogen. (b) In vitro
polysome treatment
A reaction mixture was prepared containing, in a final vol. of 25 ml: 50 n-nr-Tris*HCl buffer (pH 7*4), 125 mM-MgCl,, 80 mu-KCl, 1 mM-dithiothreitol (Sigma), 1 mM-ATP (Sigma), 0.08 mM-GTP (Sigma), 4 mM-creatine phosphate (Calbiochem) and 40 rg of creatine phosphokinase/ml (Calbiochem), 40 pg yeast tRNA/ml (General Biochemicals) and 0.02 mM of 20 amino acids (Calbiochem). The crude supernatant fraction was required for maximum incorporation activity (2.5 ml per 25ml incubation mixtures). Polysomes were added to a final concn of 25 mg/ml (750 A aao units per 25 ml). Finally the required protein synthesis inhibitor was present in t,he mixture as indicated in each case. The above reaction mixture was incubated at 30°C for 10 min, cooled to 0°C and layered on 10 ml of 60% sucrose in the standard buffer. Polysomes were spun down in a 60Ti Spinco/ Beckman rotor at 200,000 g for 4 h. They were resuspended in a small volume of standard buffer and clarified by centrifugation at low speed. The polysome concentration was determined and the preparation was immediately used for a binding assay. 75% of the initial polysomes were recovered. When polysomes were treated with puromycin, the incubation was carried out for 30 min and the released ribosomes were sedimented in the ultracentrifuge through a 20% sucrose layer.
(c) Polyaome activity (i) Amino acid incorporation
assay
The following reaction mixture was taken in a final vol. of 0.05 ml : 50 mna-Tris . HCl buffer (pH 7*4), containing 12.5 mM-Mg&, 80 m&r-KCl, 1 mM-dithiothreitol, 1 mnr-ATP, 0.08 mM-GTP, 4 mM-creatine phosphate, 40 pg creatine phosphokinase/ml, 40 rg yeast tRNA/ml and 0.02 mM of 19 amino acids (phenylalanine was omitted). [14C]Phenylalanine
452
M. BARBACID
AND
D.
VAZQUEZ
(New England Nuclear, 458 mCi/mmol) was added to a final concn of 0,015 mu, reducing its spec. act. 2.5 times. The crude supernatant fraction was added to detect a maximum of incorporation activity (usually 5 ~1). The reaction was initiated by adding 1.5 A,,, units of polysomes per tube. Incubation was at 30°C for the required time and was stopped by addition of l-5 ml cold trichloroacetic acid with 1% Casamino acids. Samples were heated for 15 min at 80 to 9O”C, cooled off, filtered through GFjC glass fibre Whatmail filters and radioactivity was determined. Va,lues in controls without polysomes wore subtracted in all cases. (ii) Peptidyl-puromyccin
a88ay
The following reaction mixture was taken in a final vol. of 0.05 ml: 50 mu-Tris .HC’l containing 125 mM-MgCl,, 80 mM-KCl, 0.5 rnivr-GTP, 5 ~1 of the crude supernatant fraction and 2.3 A,,, units of polysomes. The mixture was preincubated at 30°C for 7 min. 4 pM-[3H]pUrOmyCin (The Radiochemical Centre, Amersham, 3.7 Ci/mmol) was then added and the incubation was continued for 1 min more. The reaction was completed by addition of 1 ml of 10% cold trichloroacetic acid. Samples were filtered as previously described were used. Values (Pestka et al., 1972) except that GF/C glass fibre filters (Whatman) obtained in blank tubes, in which 10% trichloroacetic acid was added before [3H]puromycin, were subtracted in all cases. Inhibitors were added either at the beginning of the incubation (zero time) or simultaneously with puromycin (at 7 min) depending on the assay (see Results). (d) Sucrose gradient analysis Polysomes or ribosomes were analysed in 5.5-ml linear sucrose gradients of 15% to 300/, sucrose in standard buffer by centrifugation in an SW50.1 Spinco/Beckman rotor at 45,000 revs/min for 30 min and monitored in an ISCO model 183 gradient fractionator. Profiles of all the preparations described in the present work were plotted in the appropriate Figures.
(e) Quantitative
binding of [~H]ankomycin
Binding of [3H]anisomycin (260 mCi/mmol) (Barbacid & Vazquez, 1973) to ribosomcs was studied following the sedimentation method as previously described (Fernandezbuffer (pH 7*4), containing 30 m&I-MgCl, and Mufioz et al., 1971) in 20 mM-Tris.HCl 100 m&r-KC1 at 0°C. Data were plotted according to Scatchard (1949) and the dissociation constant K, is presented as a measure of the affinity of [3H]anisomycin for the ribosome. In all cases the ribosome is considered as a single unit even when it is bound t,o mRNA forming part of a polyeome.
3. Results (a) Ribosomd
heterogeneity of eukuryotic
ribosmes
A heterogeneity in standard preparations of washed eukaryotic ribosomes was found when studying the binding of [3H]anisomycin and [3H]gougerotin (Barbacid $ Vazquez, 1974a,b). The heterogeneity might be due either to differences in the functional states of the ribosomes or in their response to the high-salt treatment. In order to resolve this problem we have studied [3H]anisomycin binding to run-off ribosomes prepared from spheroplasts treated with 1 mM-sodium azide (Fig. l(a)) or incubated for ten minutes at 8°C (Fig. l(b)). Under these experimental conditions the initiation process of translation is inhibited in vivo. Therefore, polysomes are not formed and all ribosomes are free of messenger RNA. As shown in Figure l(a) and (b) these ribosomes are homogeneous for [3H]anisomycin binding. Very similar results were obtained with both types of run-off ribosome preparations. At saturation, we may take it that one molecule of
RIBOSOME
CHANGES
DURING
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453
[3H]anisomycin is bound per ribosome. The aflinity value was Ktboff = 3.5 x 10m6M for ribosomes prepared from sodium azide-treated spheroplasts and Kz-Off = 3.3 x 10m6 M for ribosomes prepared from spheroplasts incubated at 8°C. The parameters indicated above for [3H]anisomycin binding differ from those previously found with standard washed ribosomes (Barbacid & Vazquez, 1974a). Since a different strain of yeast was used before, we have now studied [3H]anisomycin
FIN. 1. Scatchard plots of the data for [sH]anisomycin binding to free ribosomes obtained from : (a) 1 mr+r-sodium azide-treated yeast spheroplasts; (b) 8°C incubated yeast spheroplasts; (c) yeast cells, following standard methods (“high-salt washed ribosomes”) (Barbacid & Vazquez, 1974a); and (d) 1 mna-sodium azide-treated yeast spheroplasts, followed by a high-salt washing treatment similar to that in (c). In all cases the binding assays were carried out by the ultracentrifugation method at 0°C (140,000 g, 2 h) under the following ionic oonditions: 60 man-Tris.HCl buffer (pH 7.4), containing 30 mM-MgCl, and 100 mM-KCl. Ribosome concentrations were 3 x 10-s M for tubes with [sH]anisomycin comma ranging from 2 x 10-r 116to 2 x 10-s M and 6 x 10-s M for tubes with [3H]vols were 80 and 60 4, anisomyoin concns ranging from 2 x 10-s M to 2.6 x 10e5 M. Incubation respectively, and samples (20 and 10 4, respectively) were taken before and after sedimentation of the ribosomes. The profiles indicate the sucrose gradient analysis (A,,,) of each ribosome or polysome preparation.
binding to standard washed ribosomes from the S. cerevisiae strain A224A, which was used throughout this work. For this purpose yeast was grown in a 20-litre fermentor, growth was stopped by addition of ice at the logarithmic phase and cells harvested by continuous centrifugation. Washed ribosomes were obtained (see Materials and Methods) and [3H]anisomycin binding was studied (Fig. l(c)). Approximately 30% of the ribosomes bind the antibiotic with a higher affinity defined by KffFW = 5.2 x 10e7 M, whereas the remaining 70% interact with a lower affinity (K@”,- = 5x 10d6 M). These results are practically identical to those obtained 30
454
M. BARBACID
AND
D.
VAZQUEZ
previously with similar ribosome preparations from X. cerewisiue strain PM-l, if the changes introduced in the ionic conditions are taken into account (see Fig. 1 in Barbacid t Vazquez, 1974a). However, the differences between the results presented in either Figure l(a) or (b) and Figure l(c) might be due to the high-salt treatment or the cell-disrupting procedure. Therefore, a ribosome preparation, identical to that used in the experiment shown in Figure l(b), was washed with 0.5 IN-NH&I and 0.1 M-MgCl,. The results obtained when [3H]anisomycin binding to these ribosomes was studied, clearly she\\ that two different types of ribosomes were obtained after high-salt treatment of run-off ribosomes (Fig. l(d)). However, in this case the percentage of each type of ribosome and the value of the dissociation constant of the higher affinity ribosomes differ considerably from those of Figure l(c) (K,,, = 14~ 10e6 and y1 = O-37: K,,, = 5.6 x lOa and v2 = 0.48. These results might indicate that the parameters of the heterogeneous binding are somehow dependent on the treatment of the cells prior to the isolation of the ribosomes. (b) Preparation and activity of polysomes It is generally accepted that ribosomes undergo conformational changes during the steps of the ribosomal cycle (Haselkorn $ Rothman-Denes, 1973, review). The ability of [3H]anisomycin to distinguish between the structure or conformation of run-off and high-salt treated ribosomes, encouraged us to study [3H]anisomycin binding to polysomes at different well-defined steps in order to detect any possible ribosomal changes. This required the preparation of polysomes without free ribosomes, which was achieved by a cycloheximide (3x 10m5 M) pre-treatment of the spheroplasts followed by centrifugation of the polysomes through a cushion of 60% sucrose. Our polysomes synthesized polypeptide chains linearly for at least 15 minutes and an average of 30 to 35 amino acids per ribosome was incorporated in 45-minute incubations (results not shown). (c) [3H]anisomycin binding to untwxzted polysomes Polysomes from control untreated spheroplasts show at least two different types of structure according to their affinity for [3H]anisomycin (Fig. 2). One third of the total polysomes interacting with the antibiotic show a high-affinity binding (K,,, = 8.1 x 10e7 M), whereas the other two-thirds of the polysomes have a lower affinity W d.L -- I.2 x 10m5 M). In this assay the [3H]anisomycin interaction with free ribosomes (approx. 15% of the total preparation) cannot be detected because the value of their dissociation constant is 3.5 x 10e6 M. Therefore, the percentage of polysomes with low affinity (K,,,) calculated from the Scatchard plot (Fig. 2) is probably higher than the real value. As shown before with other ribosome preparations, one molecule of antibiotic is bound per ribosome. (d) Peptidyl-puromycin fotiion This polysome heterogeneity might well reveal two different ribosomal stages belonging to two different steps of the elongation process in protein biosynthesis. Therefore, we developed a system for translocation and peptide bond formation based on that described by Pestka et al. (1972) with some important modifications, especially in the ionic conditions.
RIBOSOME
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466
In agreement with Pestka et al. (1972) we observed that a maximum of reactivity is found between 0.8 and 1-O ~-Kc1 almost independently of magnesium concentration. However, the crude supernatant fraction is able to stimulate the reaction as a consequence of a translocation process only at KC1 concentrations lower than 300 mM
0.6
0.8
I I.0
FICA 2. Soatohard plot of the data for [3H]anisomyain binding to untreated yeast polysomes. Data were obtainedfrom an ultrecentrifugation assay (14O,OOOg, 2 h) carried out at 0°C. 3 x 10Tg~polysomes were added for an [3H]anisomyain concn range of 0.26 to 2 x 10eB M, and 6 x 1OYp M were present for an [sH]anisomycin conan range of 2 x 1Om8 to 2.6 x 10m5 M. Ionic conditions and incubation volumes as in legend to Fig. 1. The profile shows the polysome content of t,he preparation by mesm of a suorose gradient analysis.
(unpublished results). Therefore, we used the same ionic conditions as described for amino acid incorporation by the polysomes. In all cases [3H]puromycin (4 x 1Oe6 M) was added after translocation. The effect of an inhibitor present at the beginning of the reaction reflects its effect on translocation and/or peptide bond formation. If the inhibitor was added just after the translocation preincubation, its effect on peptide bond formation only could be measured. The difference between both systems will give the effect of the inhibitor on tranalocation. Results are shown in Table 1. Diphtheria toxin, fusidic acid and cycloheximide appear to be inhibitors of translocation whereas sparsomycin completely inhibits peptide bond formation. Doxycycline affects neither reaction. These results are in agreement with the generally accepted mechanism of action of these inhibitors except in the case of fusidic acid (see Discussion) (Gale et al., 1972 ; Vazquez, 1974, reviews).
(0.012 mM) (O-01 man) (1 m4 (1 mx) (0.2 mma) (0.02 mM) 1.64 1.43 1.43 1.61 o-01
1.49
0 4 10 0 99
0
0.68 0.68 0.69 1.46 0.02
1.49 0.62
Pep?y:PM formation (pmol)
Peptidyl-transferase plus translocation
0.86 0.76 0435 0.08 -0.01
0 0.87
(pm4
Peptidyl-tRNA not translocated
99 86 75 7 0
0 100
o/0 Inhibition
Translocation
The peptidyl-transferase assay (1st column) was carried out after a prior translocation of the pept’idyl-tRNA from the A-site to the P-site by preincubation of the polysomes for 7 min at 30°C in the presence of a crude supernatant fraction and GTP. In the peptidyl-transferase plus translocation assay (2nd part of the Table), the inhibitors were present before the translocation. The 3rd part of the Table (peptidyl-tRNA blocked in the A-site) was obtained from the differences between the first and second parts, and hence the inhibition of the translocation reaction was calculated. The experimental conditions were as follows: 50 m&r-Tris.HCl buffer (pH 7+4), 12.5 mM-MgCl,, 80 mar-KCl, 0.5 m&r-GTP, 5 ~1 of a crude supernatant fraction, 2.3 Aleo units of polysomes and 4 PM-[aH]puromycin (spec. act. 3.7 Ci/mmol) in a 0.06 ml final vol. The translocation reaction was initiated with polysomes. The subsequent puromycin (PM) reaction was started by adding the labelled puromycin and incubating for a further minute also at 30°C. Reaction was stopped by the addition of 1 ml of cold 10% trichloroacetic acid and filtered through GF/C Whatman glass fibre filters as previously described (Pestka et al., 1972). The polysome preparation contained less than 15% of free ribosomes.
Complete - Supernatant + Diphtheria toxin plus NAD + Fusidio acid + Cycloheximide + Doxycycline + Sparsomycin
1
formation by translocated yeast polysomes
Peptidyl-transferase assay after translocation Peptidyl-PM formation o/0 Inhibition (pm4
Peptidyl-puromycin
TABLE
RIBOSOME
CHANGES
DURING
TRANSLATION
457
(e) [3H]oni.sonaycin binding to in vitro treated polysomes (i) Cyclohexinaide pooolyaonaes Cycloheximide stops protein synthesis, most probably aa a consequence of a single interaction with the ribosome. Hence, it was expected that all the cycloheximidetreated polysomes had been blocked in the same elongation step in protein synthesis. Our results obtained by means of the puromycin reaction suggest that cycloheximide blocks polysomes with peptidyl-tRNA bound to their A-site. This is in agreement with previous experiments by other workers (Baliga et al., 1969,197O; Obrig et al., 1971). The results obtained from studying [3H]anisomycin binding to cycloheximide(10e3 M) treated polysomes are shown in Figure 3. All the polysomes with binding activity are homogeneous, showing an atEnity for the antibiotic (KdCHX = 1.2 x lo- 5 M) identical to the low-affinity binding of untreated polysomes (K,,, = 1.2 x 10v7 M). The saturation value (J) of cycloheximide-treated polysomes was lower than that of untreated polysomes. However, similar low values of 9 were obtained with polysomes treated in vitro with other antibiotics (see results presented below) suggesting that parts of the polysomes were damaged.
Fra. 3. So&chard plot for [sH]anisomyoin binding to yeast oyoloheximide-treated polysomes. The in vitro treatment was oarried out by inoubating the polysomes for 10 min at 30°C in the presenoe of 1 mna-oyoloheximide as described in Matarials and Methods. Polysomes isolated by sedimentation through a 60% suoroae layer were immediately taken for the ultraoentrifugation binding assay. Polysome ooncentrations were 3 and 6 x 10-O I for [sH&nisomyoin oonon ranges of 1 to 3 x 10Te M and 3 x 10-s M to 3 x 10e6ar, respeotively. Otherwise the experimental conditions were as indicated in the legend to Fig. 1.
(ii) Doxycycline polysmaes In order to study the binding of anisomycin to polysomes with peptidyl-tRNA bound to the P-site, polysomes were treated in vitro with O-2 rnlcl-doxycycline for ten minutes at 30°C to inhibit aminoacyl-tRNA binding to the A-site, and therefore block peptidyl-tRNA in the P-site (Gale et al., 1972; Vazquez, 1974, reviews). As Figure 4 shows, these ribosomes were heterogeneous for [3H]anisomycin binding. However, up to 60% of the active polysome fraction bound the antibiotic with the higher affinity (Kt,: = O-82x 10ee M) which differs from the low-affinity binding of polysomes with the peptidyl-tRNA bound to the A-site.
488
M. BARBACID
AND
D. VAZQUEZ
Y
FIG. 4. Scatchard plot of d&a for [3H]anisomycin binding to yeast doxycycline-treated polysomes. The in vitro inoubation was oarried out at 30°C for 10 min in the presenoe of 0.2 mMdoxycychne. After sedimentation, polysomes were immediately taken for the following binding assay: polysome ooncentrations were 2.5 x 10eB M (for tubes with [3H]anisomycin concns ranging from 0.2 to 1 x 1O-e M), 4~ 10m3 M ([3H] anisomycin conctns from 1 x 10eB to 1 x 1Ou5 Y) and 6 x 10-s M ([3H]anisomyoin concns from 1 to 4 x 10-s M). For other experimental aonditions see legend to Fig. 1.
(iii) Diphtheria; toxilt and fmidic acid polysomes It was also of interest to study the interaction of [3H]anisomycin with polysomes carrying stably bound elongation factor EF-2. This was possible by treating polysomes in the presence of GTP and EF-2 with either diphtheria toxin + NAD or fusidic acid, which promote the formation of either ADP-ribosyl-EF-2 *GDP-ribosome or EF-2 efusidic acid* GDP-ribosome, respectively. In the first complex peptidyl-tRNA is located in the A-site. Similarly, the substrate is not reactive with puromycin in the fusidic acid-stabilized complex. Polysomes were incubated at 30°C for ten minutes in the presence of either 12 pMdiphtheria toxin plus 10e5 M-NAD or 1 rnllr-fusidic acid, and subsequently isolated by sedimentation through a 60% sucrose layer, which contained 1 mm-fusidic acid when present in the incubation mixture. With both complexes, homogeneity of [3H]anisomycin binding was observed (Fig. 5) suggesting that all the polysomeswere blocked at the same step. As with other in vitro treated polysomes, the saturation values were lower than 1, most probably due to partial inactivation during the preparation procedure. The most important feature of these experiments is the almost identical value of the dissociation constant in both cases. From Figure 5(a) an ailinity
RIBOSOME
CHANGES
DURING
TRANSLATION
459
Fm. 6. Scatchard plots of the data for [sH]anisomycin binding to polysomes pretreated with either: (a) diphtheria toxin; or (b) fusidic acid. The in vitro treatments were carried out as described in Materials and Methods in the presence of 12 PM-diphtheria toxin plus 1 x 10m5 M-NAD or 1 mM-fusidia acid. All the experimental conditions concerning the binding assay were as indiaated in the legend to Fig. 1. The polysome profiles of the preparations used for [3H]anisomycin binding are shown as in all the Figures.
of KaDT = 1.8 >: 10 - 6 M can be deduced for the interaction between [“HJanisomycin and diphtheria toxin-treated polysomes. A dissociation constant of KaFA = 1.5 x 10s6 M was found (Fig. 5(b)) in the caseof fusidic acid treatment. (iv) Puromycin-released ribosomes When polysomes were incubated for 30 minutes at 30°C in the presence of 1 mMpuromycin under conditions in which they are actively incorporating amino acids, a breakdown of polysomes into monosomes occurred. We measured the ability of those puromycin-released monosomes to interact with [3H]anisomycin in order to compare them with run-off or high-salt washed ribosomes. The Scatchard plot of Figure 6 clearly shows a homogeneity with a saturation value of 0.6. The resulting dissociation constant KdPM = 3.6 x 10e6 M is practically identical with that of in vivo run-off ribosomes (either sodium azide- or cold-treated spheroplasts, Fig. 1).
4. Discussion Our results show that conformational changes of the ribosome during the process of translation can be detected by quantitative studies of [3H]anisomycin binding. Furthermore, we have shown that high-salt washing of the ribosomes, by the standard methods used to separate the different protein factors required for the process of translation, also causes some conformational or structural changes that lead to heterogeneous ribosome populations. Previously (Barbacid BEVazquez, 1974a) we have demonstrated that these changes mainly affect the 40 S eukaryotic ribosome subunit.
460
M. BARBACID
AND
D. VAZQUEZ
FIR 6. Soatchard plot for [3H]anisomycin binding to ribosomes released from polysomes by in vitro puromyain treatment. The in vitro treatment only differed from those aarried out with other inhibitors in thet the incubation time was 30 min. Puromycin-released ribosomes were isolrtted by sedimentation in the ultraaentrifuge through 8 20% sucrose layer instead of a 60% layer as in the case of the in vitro treated polysomes. The ultraoentrifugation binding assay was aarried out identically to those described in the legend to Fig. 1.
In a simplified model of protein synthesis, ribosomes can be found in two different stages: the free ribosomes (between termination and initiation) and the polysomes (engaged in translation). The present study of [3H]anisomycin binding to these two types of ribosomes leads to the following conclusions: (1) free ribosomes (“run-off ribosomes”) are homogeneous with regard to anisomycin binding, and (2) they differ from high-salt washed ribosomes (Fig. l), and (3) from polysomes (Fig. 2), which are heterogeneous. The heterogeneity of polysomes with regard to anisomycin binding is due to at least two distinct conformational states of ribosomes during the elongation cycle. From the results obtained with [3H]anisomycin binding to cycloheximide-treated polysomes, it is clear that: (1) cycloheximide blocks the polysomes in a defined state (Fig. 3); and (2) this state, which corresponds to the phase in which the peptidyltRNA is bound to the A-site (Table I), is one of those found with untreated polysomes (low-affinity binding, K,,, = KdCHX= 12 PM). Doxycycline-treated polysomes do not show total homogeneity. This might be due to the small inhibitory effect of the tetracycline group of antibiotics on aminoacyltRNA binding in eukaryotic cell-free systems. It is suggested that the high-affinity binding defined by K,DC 1 - O-82x 10 - ’ M (up to 60% of polysomes) corresponds to the state in which peptidyl-tRNA is bound to the ribosomal P-site (Fig. 7). As this value is practically identical to K,., = 041 x 1Oms M, we can ascribe the second structure found with untreated polysomes (the high-affinity binding) to the step blocked by doxycycline. As untreated polysomes were obtained from spheroplasts rapidly cooled at O”C, it seems as if aminoacyl-tRNA binding and translocation are the most cold-sensitive steps in the elongation process of protein synthesis in yeast. It should be taken into
RIBOSOME
CHANGES
DURING
TRANSLATION
461
Diphtheria toxin or Fusidic acid
Cycloheximide
Y Termination Elongation cycle
K,,L=Kd CHx= 12/&M
z
FIG. 7. Scheme of the ribosome NaAz, sodium azide.
32
U
K,,“=K:‘=
O+M
A Doxycycline
oyole studied
by [3H]anisomyoin
binding.
PM, puromyoin;
account that variations in the ionic conditions during the polysome isolation procedure could modify the proportions of pre and post-translocated ribosomes found in our untreated polysome preparations (Fig. 2). Fusidic acid is widely accepted as an inhibitor of aminoacyl-tRNA binding in intact bacteria and in several cell-free systems (Gale et al., 1972; Vazquez, 1974). Theee results have been explained by the blocking of the ribosomal A-site by the EF-G (or EF-2) *GDP*ribosome complex which is stabilized by fusidic acid. The previously reported inhibitory etIeot of fusidic acid on translocation in model prokaryotic and eukaryotic cell-free systems has been explained on the basis of a trapping of all the available EF-G or EF-2 by the free ribosomes in the reaction mixture (Vazquez, 1974). However, the inhibition on trenslocation by fusidic acid observed in our pure polysomal system (Table 1) cannot be due to depletion of EF-2 through such a trapping effect because: (1) the fusidic acid-treated polysomes themselves seemed to interact with EF-2, as evident from their changed affinity for [3H]anisomycin upon the addition of the crude supernatant fraction in the presence of fusidic acid (Fig. 5(b), see below) ; and (2) increasing amounts of the supernatant fraction (15 times) did not reverse the inhibitory effect of fusidic acid (results not shown), contrary to the results which we obtained previously in a model ribosomal system (Vazquez, 1974). Our results from both [3H]anisomycin binding (Fig. 5) and peptidyl-transferase studies (Table 1) strongly suggest that fusidic acid and diphtheria toxin block polysomes in a very similar, if not identical, defined conformation. Both reagents, like cycloheximide, prevent functional trsnslocation as measured by puromycin reactivity of peptidyl-tRNA (Table 1). In the case of diphtheria toxin, peptidyl-tRNA remains in the A-site; in the case of fusidic acid, the position of peptidyl-tRNA has not yet been defined. Unlike cycloheximide, however, diphtheria toxin and fusidic acid confer on polysomes a high affinity to anisomycin. This is presumably due to the stable (EF-2eribosome) complex formation known to result with diphtheria toxin (Bermek, 1972) and fusidic acid (Gale et al., 1972; Vazquez, 1974). From the almost identical dissociation constants of anisomycin for diphtheria toxin- and fusicid acid-treated
462
M. BARBACID
AND
D. VAZQUEZ
polysomes (KdDT = 18x 10m6 M and K dFA = 1*5X 10e6 M), it appears that the ADP-ribosylation of EF-2 by diphtheria toxin does not modify its interaction with polysomes. Considering the above results, it would be of interest to study [3H]anisomycin binding to polysomes with peptidyl-tRNA bound to the P-site and aminoacyl-tRNA bound to the A-site. This polysomal preparation might be obtained “a priori” by freezing the polysomes with inhibitors of peptide bond formation. However, we have observed that yeast polysomes blocked with a peptide bond formation inhibitor like blasticidin S (Vazquez, 1974) behave similarly as control polysomes for [3H]anisomycin binding (results not shown). This result might be expected since peptidyl-transferase inhibitors reversibly block this ribosome centre. Therefore, when the inhibitor is not bound, peptidyl-transferase immediately catalyses peptide bond formation and polysomes remain heterogeneous in the same way as untreated polysomes. Sparsomycin, a very effective inhibitor of peptide bond formation (Table l), could not be used, since when it is bound to polysomes it strongly inhibits [3H]anisomycin binding. Indeed, with sparsomycin-treated polysomes, only 8% of the total polysome preparation bound [3H]anisomycin. The dissociation constant was found to be around 3 PM. This interaction must undoubtedly correspond to the small fraction of free ribosomes. The rest did not bind the labelled antibiotic, at least with a dissociation constant lower than 100 pM. Known specific inhibitors of eukaryotic peptide bond formation cannot be used because of their inhibitory effect on [3H]anisomycin binding (Barbacid & Vazquez, 1974u). The results discussed above can be summarized as indicated in Figure 7. The dissociation constants for [3H]anisomycin binding corresponding to the different conformational and/or structural states of the ribosomes during the process of translation are as follows: (1) free ribosomes (Kf-“’ = 3.5 PM); (2) post-translocated ribosomes (K$ or K,,, = 0.8 PM); (3) pre-translocated ribosomes (KdCHX or K,,, - 12 PM); and (4) polysomes with EF-2 bound (KdDT or .KdFA = 1.8 to 1.5 pM). It should be pointed out that the highest affinity for [3H]anisomycin binding was observed with post-translocated ribosomes (polysomes with peptidyl-tRNA bound to the P-site) which is precisely the step in which the antibiotic should be bound to the ribosome in order to exert its effect by blocking peptide bond formation. We thank Dr J. Modolell for his interest and for criticism of the manuscript. We am also grateful to Dr A. Jimenez and L. Sanchez for their advice in the polysome preparation procedure and to Miss A. Martin for expert technical assistance. This work has been supported by grants from Fondo National para el Desarrollo de la Investigation Cientifica, U.S. National Institutes of Health (A108598) and Lilly Indiana of Spain. One of us (M. B.) was the holder of a fellowship of Plan de Formation de Personal Investigador.
REFERENCES Baliga, B. S., Pronczuk, A. W. & Munro, H. N. (1969). J. Bid. Chem. 244, 4480-4489. Baliga, B. S., Cohen, S. A. & Munro, H. N. (1970). FEBS Letters, 8, 249-252. Barbacid, M. & Vazquez, D. (1973). Anal. Biochem. 56, 18-25. Barbacid, M. & Vazquez, D. (1974a). J. Mol. Biol. 84, 603-623. Barbacid, M. & Vazquez, D. (19743). Eur. J. Biochem. 44, 445-453. Bermek, E. (1972). FEBS Letters, 23, 95-99. Fernandez-Mtioz, R., Monro, R. E., Torres-Pinedo, R. & Vazquez, D. (1971). I&T. Biochern. 23. 185-193.
J.
RIBOSOME Gale, E. P., Cundliffe,
CHANGES
DURING
TRANSLATION
463
E., Reynolds, P. E., Richmond, M. H. Jz Waring, M. J. (1972). Ln of Antibiotic Action, pp. 278-379, John Wiley & Sons, London. Hartwell, L. H. (1967). J. Bactertil. 93, 1662-1670. Haselkorn, R. & Rothman-Denes, L. (1973). Alznu. Rev. Biochem. 42, 397-438. Hutchison, H. T., Hartwell, L. H. & McLaughlin, C. S. (1969). J. Bacterial. 99, 807-814. Obrig, T. G., Gulp, W. J., McKeehan, W. 1,. & Hardesty, B. (1971). J. Bid. Chew.. 246, 17P181. Pestka, S., Goorha, R., Rosenfeld, H., Neurath, C. 8: Hintikkn, H. (1972). -7. Bfiol. C!I/cvv. 247, 4258-4263. Scatchard, G. (1949). Ann. N.Y. Acad. Sci. 51, 660-672. Vazquez, I). (1974). PEBS Letter,?, 40, suppl. S63-S84. The Molecular
Basis
