[51] Ribosomal peptidyltransferase: Binding of inhibitors

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481

Siegler, u n p u b l i s h e d observations) have little or no effect. A n u m b e r o f antibiotic inhibitors o f p r o t e i n synthesis act specifically on the peptidylt r a n s f e r a s e center, a n d p r o v i d e useful tools for its study. 21'29 Distribution. P e p t i d y l t r a n s f e r a s e is i n t e g r a t e d into the s t r u c t u r e o f the larger ribosomal subunit in all species that have so f a r b e e n e x a m i n e d , i.e., E. coli, B. subtilis, a° Anacystis montana, 3° yeast, 31 protozoa, m rat liver, 33 a n d h u m a n tonsils. 3''34 T h e ribosomal p e p t i d y l t r a n s f e r a s e s in these various species are similar to one a n o t h e r in m a n y respects, but a difference between the fine structures at the catalytic centers o f 70 S a n d 80 S ribosomes ( f r o m p r o k a r y o t i c a n d eukaryotic organisms, respectively) is indicated by differences in sensitivity to antibiotics? T M 29R. E. Monro and D. Vazquez,J. Mol. Biol. 28, 161 (1967). 3°D. Vazquez, M. L. Celma, and R. E. Monro, unpublished data (1968). 31D. Vazquez, E. Battaner, R. Neth, G. Heller, and R. E. Monro, Cold spring Harbor Symp. Quant. Biol. 34, 369 (1969). raG. Cross, personal communication (1969). 33T. Staehelin and A. Falvey, personal communication (1969). 34R. Neth, R. E. Monro, G. Heller, E. Battener, and D. Vazquez, Fed. Eur. Biochem. Soc. Lett. 6, 198 (1970).

[51] Ribosomal Peptidyltransferase: Binding of Inhibitors By RAFAEL FERNANDEZ-MuI~oz,

ROBIN E. MONRO, and DAVID VAZQUEZ

Peptide b o n d f o r m a t i o n in p r o t e i n biosynthesis is catalyzed by a p e p t i d y l t r a n s f e r a s e catalytic center on the larger r i b o s o m a l subunit. 1'2 A n u m b e r o f antibiotic inhibitors o f protein synthesis specifically block the peptidyl t r a n s f e r reaction. Investigation o f the interaction o f such antibiotics with the r i b o s o m e provides a m e a n s to analyze not only their m o d e s o f action, but also the n a t u r e o f the p e p t i d y l t r a n s f e r a s e center. C h l o r a m p h e n i c o l , lincomycin, a n d a n u m b e r o f o t h e r specific 70 S inhibitors o f peptidyl t r a n s f e r bind to the r i b o s o m e at sites that are at or closely linked to the p e p t i d y l t r a n s f e r a s e c e n t e r ? "4 In the 1R. E. Monro, T. Staehelin, M. L. Celma, and D. Vazquez, Cold Spring Harbor Syrup. Quant. Biol. 34,357 (1969).

2D. Vazquez, E. Battaner, R. Neth, G. Heller, and R. E. Monro, Cold Spring Harbor. Syrup. Quant. Biol. 34, 369 (1969). 3B. Weisblum and J. Davies,Bacteriol. Rev. 32,493 (1968). 4D. Vazquez, T. Staehelin, M. L. Celma, E. Battaner, R. Fernandez-Mufioz, and R. E. Monro, in "Inhibitors: Tools in Cell Research" (T. Bucher and H. Sies, eds.), p. 100. Springer, Berlin, 1969.

482

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[51]

present paper we describe two methods for the analysis of antibioticbinding to ribosomes. The assays give estimates for the dissociation constant and number of available binding sites, and are suitable for correlation with resolved assays of the peptidyl transfer reaction and of substrate-binding at the peptidyltransferase center. The methods are applicable not o n l y to a variety of labeled compounds that bind to the ribosome, but also to unlabeled compounds that compete with the labeled compounds for binding.

Principle Two methods are described for the quantitative analysis of the binding to ribosomes of labeled compound. In both methods the extents of binding at equilibrium are determined in presence of different concentrations of the compound, and the data are used to calculate the dissociation constant and number of available binding sites. Method a. Equilibrium Dialysis. The ribosome solution is placed in a dialysis tube and immersed in a solution containing the radioactive compound until equilibrium is reached. The extent of binding to the ribosome is then determined from the difference of radioactivity inside and outside the tube. The accuracy of the method increases as the ribosome concentration is raised. It is therefore advantageous to use small volumes of concentrated ribosome solution rather than large volumes of dilute solution. The use of small volumes also increases the rate of equilibration. The method is versatile and accurate but requires large amounts of labeled compound (especially if several conditions are studied); it is laborious, and is difficult to apply in conditions where ribosomes are insoluble (as in the "fragment reaction"a). Method b. Ethanol Precipitation. This method has been designed to be applicable in the conditions of the fragment reaction I (with 33% ethanol v/v) in order to facilitate correlation with assay of the peptidyl transfer reaction. In such conditions the ribosomes are quantitatively precipitated, provided that ethanol but not methanol is used and that the temperature is below about 70.5 The extent of binding of a radioactive compound to the ribosomes at equilibrium is estimated from the fall of radioactivity in suspension when the ribosome precipitate is sedimented by low speed centrifugation. The method is rapid and requires only small amounts of material. The presence of 33% ethanol does not alter the number of available binding sites but may influence the dissociation constant. This does not hinder studies on the modes of action of inhibitors, and is actually advantageous in certain cases (see lincomycin, below). 5R. E. Monro, M. L. Celma, and D. Vazquez, Nature (London) 222, 356 (I 969).

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483

Alternative methods Equilibrium dialysis can alternatively be carried out using dialysis cells, n A variety of other methods can also be used to measure the binding of labeled compounds to ribosomes. Among these the following may be found useful in special cases: Method c. Adsorption of Ribosomes to MiUipore FiltersY This method is rapid and simple, but can be applied only when binding is very strong, and it gives no estimate for the dissociation constant. Method d. Sedimentation. Sedimentation of ribosomes in the ultracentrifuge followed by measurement of radioactivity in the pellet, s This method may be used to detect binding of compounds which interact weakly with the ribosome, since the concentration of ribosomes in the pellet is very high, and the blanks are very low owing to the small percentage of supernatant trapped in the pellet. The method requires large amounts of material, and is only semiquantitative. Method e. Membrane Ultrafiltration. Diafiltration 9 techniques are more rapid than dialysis and equally versatile. We found that the Diaflo membrane most suitable for the study of the binding of antibiotics to the ribosome is the XM-50. Ultrafiltration TM is of particular value in cases where only small quantities of ribosomes or protein are available. Reagents and Equipment Visking dialysis tubing pretreated as in ref. 6, and stored at 0o-5 ° in 0.1 mM EDTA [~4C]Chloramphenicol (10-20 Ci/mole), obtainable from the Radiochemical Centre, Amersham, England Salt-washed ribosomes or their larger subunits, prepared from E. coli or other bacteria by standard methods (see, for instance, Staehelin, this volume [45]). Nascent protein may interfere with antibiotic binding. The 50 S subunits prepared by the method of Staehelin are sufficiently clean, but unresolved ribosomes may contain considerable quantities of nascent protein. This can he removed by incubation of salt-washed ribosomes with 0.1 mM puromycin for 10 minutes at 30 °, followed by dialysis or ultracentrifugation to remove the puromycin. Buffered salts mix: Tris-HC1 buffer, pH 7.9 at 20 ° (giving pH 7.5 in the assay mix at 0°), 1.0 M in Tris nP. T. England, J. A. Huberman, T. M. Jovin, and A. Kornberg, J. Biol. Chem. 244, 3038 (1969). 7F. M. Chang, C.J. Sih, and B. Weisblum, Proc. Nat. Acad. Sci. U. S. 55, 431 (1966). 8D. Vazquez,Nature (London) 203,257 (1964). 9W. F. Blatt, S. M. Robinson, and H.J. Bixler, Anal. Biochem. 26, 151 (1968). ~°H. Paulus, Anal. Biochem. 32, 91 (1969).

484

RIBOSOME STRUCTURE AND FUNCTION

[51]

KC1, 2 M Mg acetate, 1.0 M Ethanol (absolute) Conical tubes (5-10 ml) Low speed centrifuge (refrigerated or in cold room) Scintillation fluid (Bray's Solution 11 or other mixtures are also suitable): Toluene, 67 % (v/v) Triton X- 100, 33 % (v/v) BBOT, 4 g/liter (CIBA) CAB-O-SIL, 40 g/liter (Nuclear Chicago) Procedure Method a. Equilibrium Dialysis. TM The ribosome solution is adjusted to a concentration roughly equivalent on a molar basis to twice the dissociation constant of the compound u n d e r study (2-7 mg/ml in the case of chloramphenicol and lincomycin). T h e n 0.2 ml samples are placed in dialysis bags and immersed completely in buffered salts solution containing the radioactive compound (with compounds of low specific activity or low dissocation constant, it may be necessary to use a larger volume for dialysis in order to obtain sufficient radioactivity). For a full determination, samples should be set up in triplicate and dialyzed against a range of at least 4 concentrations of the compound, extending both above and below the dissociation constant. Equilibration at 0°-5 ° takes 2-5 hours if there is rapid shaking or at least 20 hours if there is no shaking. After equilibration each dialysis bag is removed separately, gently mixed, and cut off at one end; two aliquots are r e m o v e d - o n e (50-100 /~1) for determination of radioactivity, and the other (10-20 /zl) for determination of ODze0. Radioactivity is measured in a scintillation spectrometer after mixing the sample with 3 ml of scintillation fluid. Samples for radioactivity determination are also taken from the solutions against which the samples have been dialyzed. The difference between the radioactivity inside and outside of a dialysis bag gives an estimate for the concentration of the ribosome-bound compound, while the radioactivity in the outer solution corresponds to the concentration of compound in equilibrium with the ribosome-bound compound. T h e ribosome concentration is calculated from the OD260. Several precautions should be taken. The operator should handle the dialysis tubing only while wearing gloves. Excess liquid should be wiped away from the tubing with tissue before insertion or removal 11G. A. Bray, A n a l . Biochem. I, 279 (1960). 12We thank W. Gilbert for a helpful discussion on this method.

[51]

BINDING o r INHIBITORS

485

of samples, in order to minimize dilution of the ribosomes. Evaporation should be limited by carrying out operations rapidly and preferably in a cold room. If several types of sample are dialyzed against the same solution, they can be distinguished from one another by leaving an extra length of empty tubing at the end and notching it in various combinations. Method b. Ethanol Precipitation. The assay is carried out in the same conditions as the "fragment reaction" using 33% ethanol. Higher concentrations of ethanol can also be employed, but not lower concentrations, since the ribosomes are then incompletely precipitated. Incubations are carried out in small, conical tubes at 0 °. Each incubate contains 100/zl of water, 50/zl of ethanol, 5/.Lmoles of Tris.HC1 buffer (pH 7.5), 40/zmoles of KCI, 2/zmoles of Mg acetate, ribosomes (or the larger subunit), and the radioactive compound under study (see below). The ribosome concentration should be equivalent to at least twice the dissociation constant of the compound, and in no case should it be lower than about 1 mg/ml (to avoid incomplete sedimentation). For compounds of low specific activity or low dissociation constant, the incubation volume may have to be increased in order to obtain sufficient radioactivity. Buffer and salts are conveniently added from a concentrated stock solution of the composition indicated under Reagents. The ethanol should be precooled, added last, and mixed in well. After incubation at 0 ° for 30-90 minutes, the suspension is centrifuged at about 3000 g for 20 minutes at 00-5 °. Then 0.1 ml of the supernatant is carefully removed and mixed with 3 ml of scintillation fluid, and the radioactivity is determined. The resultant value provides an estimate for the concentration of the labeled compound in free solution at equilibrium. For comparison, the total radioactivity is determined on parallel samples in which ribosomes are omitted. The difference between the total radioactivity and the radioactivity in free solution gives an estimate for the concentration of ribosome-bound compound. For a full determination samples should be in triplicate and at least 4 concentrations of the radioactive compound should be tested. The amounts of added compound should be adjusted so that the concentrations remaining in the supernatant cover a range extending both above and below the dissociation constant. Calculation of Dissociation Constant and N u m b e r of Sites Provided the binding sites for a given compound, A, are equivalent and independent, the following relationship holdsl3: laj. T. Edsall and J. Wyman, "Biophysical Chemistry," Chapter 11. Academic Press, New York, 1958.

486

RIBOSOME STRUCTURE AND FUNCTION

lA---i= kA. -- kA;

[51] (1)

where ~ is the number of molecules bound per ribosome, [A] is the concentration of free A, n is the number of sites per ribosome, and KA is the association constant for A. A Scatchard-type plot of v v. v/[A] is then linear with an intercept at n on the v axis, and a slope of-KA. If there is more than one class of binding site, the plot will be nonlinear, la

Sources of Error The Donnan effect is insignificant in the equilibrium dialysis assay at the salt concentrations normally employed, but might cause errors in the ethanol precipitation assay. In the latter assay there may also be errors resulting from the sedimentation of reversibly interacting components. ~4 Experimental results show that such errors are within tolerable limits for many purposes. Thus, the Scatchard plot for chloramphenicol binding approximates to linearity, and the estimate for the number of available binding sites is in agreement with the value obtained by equilibrium dialysis (see below). The interaction of some antibiotics with ribosomes may be relatively slow and not readily reversible. There may also be weak nonspecific interactions in addition to specific interaction. We have observed neither of these divergencies from ideal behavior for chloramphenicol or lincomycin. However, both effects may be significant in the case of erythromycin, ls-'7 The rate of binding can be examined by varying the time or temperature. The reversibility can be studied after binding of a radioactive ligand has taken place either by removal of the free ligand (by further dialysis or resuspension in fresh buffer) or by addition of excess unlabeled lingand? 7 Nonlinear Scatchard plots are obtained if the preparation of labeled ligand contains radioactive impurities that do not bind. Such impurities can be estimated by determination of the percentage of added radioactivity which is bound at saturating ribosome concentration. 17

Application to Chloramphenicol and Lincomycin Binding As an example of the application of the ethanol precipitation method, Fig. 1 shows a plot of data from a typical assay for the binding of [~4C] 14H. K. Schachman, "Ultracentrifugation in Biochemistry," Chapter, IV-9. Academic Press, New York, 1959. 15N. L. Oleinick and J. W. Corcoran,J. Biol. Chem. 244, 727 (1969). t6j. C. H. Mao and M. Putterman,J. Mol. Biol. 44, 347 (1969). 17R. Fernandez-Mufioz, R. E. Monro, and D. Vazquez, Fed. Eur. Biochem. Soc. Lett. in press.

[51]

BINDING OF INHIBITORS

\ .--.

%

487

.,,,.

0.5 x

0

I

0

05

I 1.0

FIG. 1. Scatchard plot of data for chloramphenicol binding from an ethanol precipitation assay. Conditions were as described in text. The ribosome concentration was 10 mg/ml, and the concentrations of added chloramphenicol were 0.66, 1.0, 1.33, 2.0, 3.3, and 6.6/zM. Data are from R. Fernandez-Mufioz, R. E. Monro, and D. Vazquez, Fed. Eur. Biochem. Soc. Lett. in press. chloramphenicol. T h e plot is linear, within e x p e r i m e n t a l e r r o r , and extrapolates t h r o u g h a point on the v axis c o r r e s p o n d i n g to about one binding site per ribosome. T h e slope c o r r e s p o n d s to a dissociation constant o f about 1.1 /zM. Equilibrium dialysis also gives an estimate o f one binding site per ribosome for chloramphenicol, and an estimate o f 0.8/zM for KCM in presence o f 10 mM T r i s . H C l buffer (pH 7.4), 100 m M NH4CI, 10 m M Mg acetate, and 5 m M fl-mercaptoethanol. 17 T h e ethanol precipitation assay has also been applied to lincomycin binding, 17 and gives an estimate o f about one binding site per ribosome and a value o f about 1/zM for KLM. T h e equilibrium dialysis assay c a n n o t be applied to the estimation o f lincomycin-binding to E. coli ribosomes because KLM in absence o f alcohol is greater than 10 /zM, and binding is hardly detectable at 10 mg/ml o f ribosomes? 7 T h e birtding o f lincomycin in absence o f alcohol can, however, be estimated by M e t h o d d (i.e., ultracentrifugation). T h e action o f alcohol on lincomycin binding may be related to its action in p r o m o t i n g the catalysis o f peptidyl transfer by ribosomes. 1 T h e binding o f a n u m b e r o f o t h e r antibiotics to E. coli ribosomes may also be p r o m o t e d by alcohol, lz Equilibrium dialysis can be applied to lincomycin binding when ribosomes f r o m gram

488

RIBOSOME STRUCTURE AND FUNCTION

[51]

positive bacteria are used: such organisms are more sensitive to the antibiotic than gram-negative bacteria, and their ribosomes have a correspondingly lower KLM(unpublished data).

Binding of Unlabeled Compounds The binding of an unlabeled compound to ribosomes can be studied by the present methods provided that it competes for binding with a suitable labeled compound. Thus, if two compounds, A and B, both react reversibly with the same set of sites, the following relationship hold#3:

=_, {KAK'A

where KA and KB are the dissociation constants for A and B, and K'A is the apparent association constant for A at a given free concentration, [B], of B. Constancy of KB over a range of [B] can be taken as strong evidence that A and B bind reversibly at mutually exclusive sites, lr This is not the case ifKB varies with [B], and Eq. 2 is then inapplicable. Both of the present methods can be used to study competition, but it is difficult to evaluate KB by the alcohol method if Ks ~< KA, because [B] cannot be directly measured and will be affected by binding of B to the ribosomes. If KB "> KA then [B] will be greater than the concentration of bound B, and [B] can be assumed to be the same as the total concentration of B. For examples of the application of competition techniques see Fernandez-Mufioz et a U 7 Range o f Application The alcohol precipitation assay is intended primarily for the correlation of studies on the binding of inhibitors with studies on the peptidyl transfer reaction, but it is also useful for examination of competition between different compounds, and for routine assay of ribosome activities. T h e equilibrium dialysis assay is of more general applicability, and can be used in a wide range of conditions. Both methods are applicable to eukaryote as well as prokaryote ribosomes. The methods are suitable for binding studies on a variety of labeled compounds, and also unlabeled compounds which compete with labeled compounds for binding. If the dissociation constant of a labeled compound is greater than about 10 mM, the assays are not readily applicable because excessive amounts of ribosomes are required. In such cases ultracentrifugation (Method d) can be used, or the ethanol precipitation method can be made more sensitive by measurement o f radioactivity in the pellet rather than in the supernatant (allowance should be made for quenching

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of radioactivity by the ribosomes). However, the methods are then only semiquantitative owing to the undetermined and variable amount of supernatant trapped in the pellets. The interaction between ribosomes and compounds which bind only weakly can often best be studied by competition with labeled compounds. 17 Modifications of the alcohol method have been applied to the study of substrate-binding at the peptidyl transferase centre, TM and to the action of sparsomycin on substrate-binding? A different approach to the study of substrate-binding at the peptidyl transferase centre has also been reported? 9 These methods have provided information on relationships between the antibiotic and the substrate binding-sites.

Properties Stability. The capacity of E. coil ribosomes for the binding of chloramphenicol, lincomycin, and related antibiotics has similar stability characteristics to the capacity for catalyzing peptidyl transfer (R. E. Monro, this volume [50]). The necessity of reactivating ribosomes after exposure to absence of monovalent cations z° should be noted. Distribution and Specificity. Peptidyl transfer is catalyzed by the larger ribosomal subunit in all species so far tested, including bacteria, yeast, protozoa, rat liver, and human tonsils. 1"2 In E. coli evidence has been reported that there is one peptidyltransferase center per ribosome, and it is reasonable to suppose that this also holds for higher organisms. Chloramphenicol, lincomycin, and a number of other antibiotics specifically inhibit the peptidyltransferase of 70 S ribosomes. They have no effect on the activities of 80 S ribosomes, and they bind to 70 S but not 80 S ribosomes? "4 These antibiotics bind at closely related sites on the 50 S subunit of the 70 S ribosomes as shown by competition studies? "4"a¢They do not bind to 30 S subunits. Anisomycin is a specific inhibitor of the peptidyltransferase of 80 S ribosomes, zal It is probable that anisomycin binds to 80 S but not 70 S ribosomes, but this has not been tested at the time of writing. A number of other inhibitors of peptidyltransferase, including sparsomycin, amicetin, and gougerotin, are active against both 70 S and 80 S ribosomes. These antibiotics do not appear to compete with the 70 Sspecific antibiotics for binding, 4 and further knowledge of their interactions must await their availability in radioactive form. Indirect evi-

lSM.L. Celma,R. E. Monro,and D. Vazquez,Fed. Eur. Biochem. 8oc. Lett. 6, 273 (1970). 1~S.Pestka,Proe. Nat.Acad. Sd. U. $. 64, 709 (1969). 2°R.Miskin,A. Zamir,and D, Elson,Biochem. Biophys. Res. Commun. 33, 551 (1968). 21R. Neth, R. E. Monro, G. Heller, E. Battaner, and D. Vazquez, Fed. Eur. Biockem. Soe. Lett. 6, 198 (1970).

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[52]

dence suggests that sparsomycin binds quite strongly to the larger ribosomal subunit if a suitably oriented peptidyl donor substrate is present at the peptidyltransferase center. 5

[52] Ribosome Peptidyltransferase By MAX E. GOTTESMAN1 Principle. The assay measures the reaction of polylysyl-tRNA with puromycin (Assay I) or with lysyl-tRNA (Assay II) in the presence of purified ribosomes and polyadenylic acid.

Assay Reagents - Assay I

Reaction mixture, 1.0 ml contains: Tris-HC1 buffer, 1.0M, p H 7.4, 0.05 ml NH4CI, 2.0 M, 0.08 ml 2-Mercaptoethanol, 0.2 M, 0.06 ml MgCI2, 1.0M, 0.01 ml Lysine, 10 raM, 0.04 ml Poly (A), 2 3 mg/ml, 0.04 ml Ribosomes, 12 mg/ml, 0.04 ml [14C]Polylysyl-tRNA, ca. 100,000 cpm/ml (specific activity, ca. 1 × 109cpm//.tmole), 0.04 ml Puromycin, s 0.46 mM (9.35 ODin5 per ml), 0.10 ml Reagents - Assay H

T h e reaction mixture is identical to that for Assay I except that unlabeled polylysyl-tRNA, 0.25 raM, 0.10 ml replaces the labeled substrate, and [3H]lysyl-tRNA* 2 × 106 cpm/ml (specific activity, ca. 1 × 109 cpm//~mole), 0.10 ml, is used in place of puromycin. Differential labeling of the polylysyl-tRNA and the lysyl-tRNA is useful for analyzing the reaction products. IM. E. Gottesman,J. Biol. Chem. 242, 5564 (1967). 2Poly A (Miles Laboratories) is dialyzed against ca. 0.1 mM EDTA and then against H20. It is stored at --20 ° in dilute Tris.HC1, pH 7.4, at an OD2e0 of 45.

3Lederle. *Stripped E. coli B tRNA (General Biochemicals)is charged with [3H]lysine(New England Nuclear) using a lyophilizedE. coli B supernatant enzyme [Fraction I of M. E. Gottesman, Canellakis and Canellakis, Biochim. Biophys. Acta 61, 34 (1962)], and is ~solatedaccording to the method of T. W. Conway[Broc. Nat. Acad. Sci. U. S. 51, 1216(1964)].