- Email: [email protected]
Inhibitory action of erythromycin on protein biosynthesis by isolated polyribosomes
ARCHIVES OFBIOCHEMISTRYANDBIOPHYSICS Vol. 214, No. 2, April 1, pp. 846-849, 1982
COMMUNICATION Inhibitory Action of Erythromycin
TADAHIKO Department
on Protein Biosynthesis
OTAKA
AND
by Isolated Polyribosomes
AKIRA KAJI’
of Microbiology, School of Medicine, Philadelphia, Pennsylvania
University 19104
of Pennsylvania,
Received August 14, 1981, and in revised form January
22, 1982
Consistent with the previous work by Pestka (Antimicrob. Agents Chemother. 5,255, 1974) on the binding of erythromycin to polyribosomes, we found that erythromycin does not inhibit protein synthesis catalyzed by polyribosomes. This is due to the presence of nascent peptidyl tRNA on the naturally occurring polyribosomes. In a soluble extract from E. coli pretreated to remove the ribosome releasing factor, polyribosomes without nascent polypeptides remain intact and can catalyze protein synthesis in the absence of initiation. In this system erythromycin effectively inhibited protein synthesis. The inhibition by erythromycin was caused by premature release of oligopeptidyl tRNA from polyribosomes.
The mode of action of erythromycin has been the subject of studies by several laboratories. It has been suggested that it is an inhibitor of peptide bond formation (1, 2), or an inhibitor of translocation (3-5). In our preceding studies using an artificial polypeptide synthesis system programmed by synthetic polynucleotide, the model system, it was suggested that it exerts its inhibitory action by releasing aminoacyltRNA or oligopeptidyl tRNA from the ribosome (6). Despite these extensive studies on the mode of action and on other aspects of erythromycin action (7 lo), erythromycin has never been shown to inhibit in vitro protein synthesis by naturally occurring polyribosomes (11-15). Since in vitro protein synthesis by naturally occurring polysomes represents an in vitro system closest to the in vivo protein synthesis system, the inability of erythromycin to inhibit protein synthesis in this system casts some doubt on the validity of the proposed mechanism of the action of this antibiotic which was derived through studies on the model system programmed by synthetic polynucleotides. It is very important therefore to demonstrate that protein synthesis by polysomes can be inhibited by erythromycin. In this communication we show that the inability i Author addressed.
to whom
all correspondence
0003-9861/82/040846-04$02.00/O Copyright All rights
0 1982 by Academic Press. Inc. of reproduction in any form reserved.
should
of erythromycin to inhibit protein synthesis by polysomes is not due to an intrinsic characteristic of the polysomes themselves. It is due to the presence of nascent polypeptides which may prevent the interaction between the antibiotic and polysomes. In addition, we demonstrate that the proposed mechanism (3, 6) by which erythromycin inhibits protein synthesis is also true on polyribosomes. MATERIALS
AND METHODS
All the materials used in these studies were the same as those used in the preceding publication (6). The reaction mixture (0.2 ml) for protein synthesis by polysomes contained 20 mM Tris-HCl (pH 7.8), 50 mM NH&l, 8 mM Mg acetate, 1 mM DTT,’ 0.2 mM GTP, 7 mM phosphoenolpyruvate, 8.9 pg of pyruvate kinase, 280 pg of E. coli soluble enzymes free of RR factor (16), 115 pg of tRNA containing 8.6 X lo4 cpm of 14C-valyl-, ‘%-alanyl-, “C-seryl-, and ‘%-glycyltRNA (other aminoacyl-tRNAs were made with ‘*Camino acids), and 0.34A~ nm unit (absorbancy at 260 nm wavelength) of intact polysomes or 0.80&60 nm unit of puromycin-pretreated polysomes (16). 2 Abbreviations used: DTT, dithiothreitol; TCA, trichloroacetic acid; RR factor, ribosome releasing factor.
be
846
ERYTHROMYCIN
ACTION TABLE
ON POLYRIBOSOME-CATALYZED
I
EFFECTOFERYTHROMYCINA ONPROTEIN SYNTHESISINTHEPOLYSOMALSYSTEM
Character of polysomes Intact Intact Intact Puromycin Puromycin Puromycin
Erythromycin A (M)
Radioactivity in hot TCA-insoluble materials (cpm) 15 min
30 min
3501 3202 3257 1431 472 429
3977 3417 3404 1925 529 479
-
treated treated treated
Note. The experimental in the text.
1om5 10m4 10-j 1om4 conditions
are described
PROTEIN
BIOSYNTHESIS
847
collected beginning at the top of the tube into 19 separate tubes after recording the optical density at 254 nm with an ISCO monitor. The three fractions from the top of the gradient were pooled and 2 vol of cold alcohol were added to this pooled solution to precipitate RNA. The precipitate was isolated, dissolved in water, and subjected to hydrolysis with 0.24 N KOH for 15 min at 36°C. After neutralizing with HCl, the solution (0.19 ml) was placed on a Sephadex G-15 column (1.3 X 38 cm) which had been equilibrated with 0.02% NaNa solution to prevent bacterial growth. The column was eluted with 0.02% NaN3 solution at the rate of 1 ml/6 min. Approximately 0.35 ml was collected for each fraction and the radioactivity of each fraction was measured in 5 ml of Bray’s solution. The radioactivity of the polypeptide (in the void volume) and that of the oligopeptide (eluted before free amino acid) was measured. Further identification of the oligopeptidyl tRNA that were released was performed as described previously (6). RESULTS
Where indicated, erythromycin A was added. After incubating for 15 and 30 min at 3O”C, the amount of radioactive protein that was insoluble in hot (95’C) TCA in 80 ~1 of the mixture was measured by the filter disk method. For analysis of sedimentation behavior of polysomes, the mixture (0.35 ml) for protein synthesis was prepared essentially the same way as described above except that it contained 6 mM Mg acetate, 26 fig of RR factor free soluble enzymes, 1.37Azo, nm unit of polysomes without nascent peptides (16), and 230 pg of tRNA mixture containing 1.7 X lo5 cpm of ‘“C-valyl-, *4C-alanyl-, i4C-seryl-, and i4C-glycyl-tRNA (other aminoacyl-tRNAs were made with “C-amino acids). Where indicated, erythromycin A or sparsomycin was added. After incubating for 20 min at 30°C the mixture (300 ~1) was placed on 4.8 ml of a sucrose gradient (15-30% in 10 mM Tris-HCl (pH 7.4), 10 mM Mg acetate, 50 mM NH&l, and 1 mM DTT). The tubes were centrifuged for 55 min at 38,000 rpm in a SW 50.1 rotor. The sedimentation behavior of polysomes was analyzed with an ISCO densitometer and fractions were collected beginning at the top of the tube. The amount of radioactive protein that was insoluble in hot (95°C) TCA in 0.1 ml of each fraction was measured by the filter disk method. For analysis of the materials released from polysomes, the reaction mixture (0.35 ml) for protein synthesis was prepared essentially the same way as described in the preceding section except that it contained 510 fig of tRNA containing 2.1 X 10 cpm of i4C-valyl- and 19 “C-aminoacyl-tRNAs and 1.77Am nm units of puromycin-pretreated polysomes. After incubating for 30 min at 3O”C, the mixture was subjected to the sucrose density gradient centrifugation as described in the preceding section. Fractions were
Inhibitory effect of erythrmycin on protein synthesis by puromycin-treated polysomes. As shown in Table I, we first confirmed previously reported observations that erythromycin does not inhibit in vitro protein synthesis by naturally occurring polyribosomes (14). It appeared likely that the inability of erythromycin to inhibit protein synthesis in this system may be caused by steric hindrance exerted by the nascent peptidyl tRNA bound to ribosomes. If this is so, polyribosomes which have lost their peptidy1 group should be sensitive to this antibiotic, It should be pointed out that polyribosomes which have lost their nascent peptidyl group ordinarily undergo rapid conversion to monosomes in crude extract because the ribosome releasing factor (RR factor) releases such ribosomes from mRNA very rapidly (16, 17). Therefore, one would not be able to conduct in vitro protein biosynthesis by such polyribosomes. However, we have developed a method to remove only RR factor from a crude soluble fraction of E. coli (18), leaving all the other necessary components for protein synthesis. With this system, it is possible to study protein biosynthesis by polyribosomes which have been pretreated with puromycin to remove nascent polypeptides. These puromycintreated polyribosomes are fairly stable in the absence of RR factor and are able to produce polypeptides without going through the regular initiation steps. As shown in Table I, erythromycin exerts a very significant inhibitory action on the polyribosomes pretreated with puromycin. As low as lo-” M erythromycin A significantly inhibited incorporation of amino acids into polypeptides. These results snow that it is the presence of a long nascent polypeptide which inhibits the action of erythromycin.
OTAKA AND KAJI
848
Release of oligwpeptidyl tRNA from polysomes b erythromycin. In as much as erythromycin was found
to inhibit protein synthesis by polysomes, it was of interest to examine whether the mode of action proposed for the model system programmed by synthetic oligonucleotides holds for the naturally occurring polysomes. As shown in Table II, this is indeed the case. The amount of oligopeptidyl tRNA released from ribosomes in the presence of erythromycin was at least five times more than the control experiment. We conclude that, in the case of polyribosomes, erythromycin inhibits polypeptide synthesis by releasing oligopeptidyl tRNA from ribosomes. Release of ribawmes from mRNA after loss of oligopeptidyl tRNA. After erythromycin releases oli-
gopeptidyl tRNA from polysomes (Table II) the remaining ribosome-mRNA complex should not have the unesterified tRNA. This complex without tRNA is expected to be more unstable than polysomes with tRNA. Consistent with this expectation, erythromycin-treated polysomes are converted to monosomes rapidly even in the absence of RR factor (Fig. 1). In the control experiment, more polysomes remained (more than 50% of total ribosomes remained as polysomes in control while only 31% remained in the presence of erythromycin) even though the polysomes incorporated amino acids into polypeptides. This is in strong contrast to sparsomycin, a peptidyl transferase inhibitor, which inhibits amino acid incorporation without converting the polysomes to monosomes (Fig. Id).
TABLE II RELEASE OF OLIGOPEPTIDYL tRNA FROM POLYSOMES IN THE PRESENCE OF ERYTHROMYCIN A
Released peptidyl tRNA (cpm)
Antibiotic Erythromycin A (lo-5 M)
Total hot TCAinsoluble cpm
Polypeptide
Oligopeptide
9888
1111
175
2752
582
864
Note. The reaction mixture (0.35 ml) for synthesis of oligopeptide and polypeptides contained 20 mM Tris-HCl (pH 7.8), 50 ZUM NH&l, 8 mM Mg acetate, 1 mM DTT, 0.2 mM GTP, ‘7 mM phospboenolpyruvate, 8.9 ~g of pyruvate kinase, 280 pg E. co& soluble enzymes free of RR factor, 510 pg of tRNA containing 2.1 x lo5 cpm of ‘“C-valyl-tRNA, and 19 ‘*C-amino acyl-tRNA, and 0.80A2Banm unit of puromycinpretreated polysomes. After incubating for 30 min at 30% sucrose density gradient centrifugation was performed. The released peptidyl tRNA was isolated from the top of gradient. It was further separated into polypeptides and oligopeptide by Sephadex G-15 as described in the text.
0
5
IO
15 200 FRACTION
5
IO
15
20
NUMBER
FIG. 1. Sedimentation behavior of polysomes after incorporation of ‘%-amino acids in vitro. Effect of erythromycin A and sparsomycin. Puromycin pretreated polyribosomes were incubated with %-amino acids under the experimental condition described in the text. (a) No antibiotic and no incubation; (b) no antibiotic was added; (c) 10e4M erythromycin A was added; (d) 10e4 M sparsomycin was added. a, Hot TCA-insoluble radioactivity in each fraction; * . *, absorption at 254 nm. Sedimentation was from right to left.
DISCUSSION The data presented in this communication indicate that after removal of nascent peptidyl group from polyribosomes, one can observe the strong inhibitory effect of erythromycin on polysome-catalyzed amino acid incorporation. This is consistent with the observation of Pestka (14) that erythromycin can not bind to naturally occurring polyribosomes unless the peptidyl group is first removed. This is also in agreement with the observation that erythromycin binds to isolated ribosomes (21, 22). What is new in this report is that after removal of the peptidyl group, one can study the protein synthesis catalyzed by these pretreated polysomes. Ordinarily, upon removal of the peptidyl group, polyribosomes undergo rapid degradation into monosomes because of the presence of RR factor (16-20, 23). In the absence of RR factor, the puromycin-treated polysomes retain their structure and catalyze protein synthesis without the normal initiation steps. It is in this system, that we were able to demonstrate the significant inhibitory effect of erythromycin on polysome-catalyzed protein synthesis. Since erythromycin is shown to inhibit the polyribosome system in vitro, one can extend the present knowledge of the mechanism of erythromycin action into the in viva action. We postulate that erythromycin, in growing bacteria, binds to 50 S ribosome subunits (24, 25) of only polysomes with oligopeptidyl tRNA or no peptidyl tRNA. Once the nascent oligopeptides reach a certain size, eryth-
ERYTHROMYCIN
ACTION ON POLYRIBOSOME-CATALYZED
romycin can not stop further elongation of that chain and protein synthesis goes on until that ribosome completes translation of one cistron. In a sense, therefore, erythromycin acts as if it is an initiation inhibitor because it only binds to ribosomes with either no peptidyl tRNA or short peptidyl tRNA. This is consistent with the evidence, put forward by Tai et al. (ll), which states that erythromycin acts during the steps which closely follow the initiation step. The mode of action of erythromycin on polysomes appears to be similar to that in the model system programmed by synthetic polynucleotides. It releases oligopeptidyl tRNA from ribosomes. In a sense, erythromycin acts like puromycin which causes premature release of nascent polypeptides (26, 27). Erythromycin is different from puromycin in two important aspects, however: Erythromycin releases oligopeptidyl tRNA while puromycin releases the peptidyl group as peptidyl puromycin; puromycin releases all peptidyl groups regardless of their size while erythromycin releases only the oligopeptidyl group. This mode of action of erythromycin is consistent with the reported observation that erythromycin stimulates accumulation of oligopeptidyl tRNA in &JO in the absence of peptidyl tRNA hydrolase (28, 29). Since peptidyl tRNA is lethal to E. coli, erythromycin enhances the lethal effect of high temperature in the mutant having temperature-sensitive peptidyl tRNA hydrolase. These considerations render further support to the unified hypothesis (6) that erythromycin binds to the ribosomal site near the peptidyl transferase, resulting in inhibition of translocation (3, 4), and peptide bond formation (1, 2), while it stimulates the release of oligopeptidyl tRNA (6). Elucidation of the exact site of binding of this antibiotic to ribosomes (30, 31) would contribute toward further understanding of the mechanisms through which ribosomes carry out peptide bond formation and translocation. ACKNOWLEDGMENTS This work was supported by USPHS Grant GM 12053. The authors appreciate the help of Steven Vaupel in the preparation of this manuscript. REFERENCES 1. MAO, J. C.-H., AND ROBISHAW,E. E. (1972) Biochemistry
11, 4864-4872.
2. MAO, J.C.-H., AND ROBISHAW,E. E. (1971) Biochemistry
10, 2054-2061.
3. TANAKA, S., OTAKA, T., AND KAJI, A. (1973) Biochim.
Biophys. Acta 331,128-140.
4. IGARASHI,K., ISHITSUKA,H., AND KAJI, A. (1969) Biochem
Biophys. Res. Commun. 37,499-504.
5. CUNDLIFFE,E., AND MCQUILLEN,K. (1967) J. MoL BioL 30, 137-146.
PROTEIN BIOSYNTHESIS
849
6. OTAKA, T., AND KAJI, A. (1975) Proc. Nat. Acad Sci. USA 72,2649-2652.
7. MAJER, J. (1981) Antimicrob.
Agents Chemother.
19, 628-633.
8. FUJISAWA,Y., AND WEISBLUM,B. (1981) J. Bacteriol
146, 621.
9. HIROCHIKA, H. (1980) Mol. Gen. Genet. 179, 581588. 10. HORINOUCHI,S., AND WEISBLUM,B. (1980) Proc. Nat. Acad Sci. USA 77, 7079.
11. TAI, P. C., WALLACE,B. J., ANDDAVIS, B. D. (1974) Biochemistry 13,4653-4659. 12. MAO, J.C.-H., AND PUTERMAN,M. (1968) J. Bacteriol. W&1111-1117.
13. OLEINICK, N. L., AND CORCORAN,J. W. (1970) International Congress on Chemotherapy, 6th, Tokyo (Proc. I; pp. 202-ZOS),University Park, Baltimore, Md. 14. PESTKA, S. (1974) Antimicrob. Agents Chemother. 5, 255. 15. PESTKA, S. (1972) J. BioL Chem. 247.4669-467s. 16. HIRASHIMA, A., ANDKAJI, A. (1973) J. BioL Chem. 248, 7580-7587.
17. HIRASHIMA, A., ANDKAJI, A. (1972) Biochemistry 11, 4037-4044.
18. OGAWA,K., ANDKAJI, A. (1975) Biochim. Biophys. Acta 402, 288-296.
19. RYOJI, M., KARPEN, J. W., AND KAJI, A. (1981) J. BioL Chem. 256,5798.
20. RYOJI,M., BERLAND,R., ANDKAJI, A. (1981) Proc. Nat. Acad. Sci. USA 78, 5973.
21. TANAKA, K., TERAOKA, H., NAGIRA, T., AND TAMAKI, M. (1966) Biochim. Biophys. Acta 123, 435-437. 22. PESTKA,S. (1974) Antimicrob. Agents Chemother. 6, 474-478. 23. KUNG, H., TREADWELL,B. V., SPEARS,C., TAI, P, AND WEISSBACH,H. (1977) Proc. Natl. Acad Sci. USA 74,3217-3221. 24. WILHELM, J. M., ANDCORCORAN, J. W. (1967) Biochemistry 6, 2578.
25. FERNANDEZ-MUNOZ,R., MONRO,R. E., TORRES, PINEDO, R., AND VAZQUEZ,D. (1971) Eur. J. Biochem. 23, 185.
26. ALLEN, D. W., ANDZAMECNIK,P. C. (1962)B&him. Biophys. Acta 55, 865.
27. YARMOLINSKY,M., AND DE LA HABA (1959) Proc. Nat. Acad. Sci USA 45.1721-1729.
28. MENNINGER,J. R. (1976) J. BioL Chem. 251,33923398.
29. MENNINGER,J. R. (1979) J BacterioL
137, 694-
696.
30. LANGLOIS,R., LEE, C. C., CANTOR,C. R., VINCE, R., ANDPESTKA,S. (1976) .I MoL BioL 106,297313. 31. LANGLOIS, R., CANTOR, C. R., VINCE, R., AND PESTKA, S. (1977) Biochemistry 16,2349-2356.
COMMUNICATION Inhibitory Action of Erythromycin
TADAHIKO Department
on Protein Biosynthesis
OTAKA
AND
by Isolated Polyribosomes
AKIRA KAJI’
of Microbiology, School of Medicine, Philadelphia, Pennsylvania
University 19104
of Pennsylvania,
Received August 14, 1981, and in revised form January
22, 1982
Consistent with the previous work by Pestka (Antimicrob. Agents Chemother. 5,255, 1974) on the binding of erythromycin to polyribosomes, we found that erythromycin does not inhibit protein synthesis catalyzed by polyribosomes. This is due to the presence of nascent peptidyl tRNA on the naturally occurring polyribosomes. In a soluble extract from E. coli pretreated to remove the ribosome releasing factor, polyribosomes without nascent polypeptides remain intact and can catalyze protein synthesis in the absence of initiation. In this system erythromycin effectively inhibited protein synthesis. The inhibition by erythromycin was caused by premature release of oligopeptidyl tRNA from polyribosomes.
The mode of action of erythromycin has been the subject of studies by several laboratories. It has been suggested that it is an inhibitor of peptide bond formation (1, 2), or an inhibitor of translocation (3-5). In our preceding studies using an artificial polypeptide synthesis system programmed by synthetic polynucleotide, the model system, it was suggested that it exerts its inhibitory action by releasing aminoacyltRNA or oligopeptidyl tRNA from the ribosome (6). Despite these extensive studies on the mode of action and on other aspects of erythromycin action (7 lo), erythromycin has never been shown to inhibit in vitro protein synthesis by naturally occurring polyribosomes (11-15). Since in vitro protein synthesis by naturally occurring polysomes represents an in vitro system closest to the in vivo protein synthesis system, the inability of erythromycin to inhibit protein synthesis in this system casts some doubt on the validity of the proposed mechanism of the action of this antibiotic which was derived through studies on the model system programmed by synthetic polynucleotides. It is very important therefore to demonstrate that protein synthesis by polysomes can be inhibited by erythromycin. In this communication we show that the inability i Author addressed.
to whom
all correspondence
0003-9861/82/040846-04$02.00/O Copyright All rights
0 1982 by Academic Press. Inc. of reproduction in any form reserved.
should
of erythromycin to inhibit protein synthesis by polysomes is not due to an intrinsic characteristic of the polysomes themselves. It is due to the presence of nascent polypeptides which may prevent the interaction between the antibiotic and polysomes. In addition, we demonstrate that the proposed mechanism (3, 6) by which erythromycin inhibits protein synthesis is also true on polyribosomes. MATERIALS
AND METHODS
All the materials used in these studies were the same as those used in the preceding publication (6). The reaction mixture (0.2 ml) for protein synthesis by polysomes contained 20 mM Tris-HCl (pH 7.8), 50 mM NH&l, 8 mM Mg acetate, 1 mM DTT,’ 0.2 mM GTP, 7 mM phosphoenolpyruvate, 8.9 pg of pyruvate kinase, 280 pg of E. coli soluble enzymes free of RR factor (16), 115 pg of tRNA containing 8.6 X lo4 cpm of 14C-valyl-, ‘%-alanyl-, “C-seryl-, and ‘%-glycyltRNA (other aminoacyl-tRNAs were made with ‘*Camino acids), and 0.34A~ nm unit (absorbancy at 260 nm wavelength) of intact polysomes or 0.80&60 nm unit of puromycin-pretreated polysomes (16). 2 Abbreviations used: DTT, dithiothreitol; TCA, trichloroacetic acid; RR factor, ribosome releasing factor.
be
846
ERYTHROMYCIN
ACTION TABLE
ON POLYRIBOSOME-CATALYZED
I
EFFECTOFERYTHROMYCINA ONPROTEIN SYNTHESISINTHEPOLYSOMALSYSTEM
Character of polysomes Intact Intact Intact Puromycin Puromycin Puromycin
Erythromycin A (M)
Radioactivity in hot TCA-insoluble materials (cpm) 15 min
30 min
3501 3202 3257 1431 472 429
3977 3417 3404 1925 529 479
-
treated treated treated
Note. The experimental in the text.
1om5 10m4 10-j 1om4 conditions
are described
PROTEIN
BIOSYNTHESIS
847
collected beginning at the top of the tube into 19 separate tubes after recording the optical density at 254 nm with an ISCO monitor. The three fractions from the top of the gradient were pooled and 2 vol of cold alcohol were added to this pooled solution to precipitate RNA. The precipitate was isolated, dissolved in water, and subjected to hydrolysis with 0.24 N KOH for 15 min at 36°C. After neutralizing with HCl, the solution (0.19 ml) was placed on a Sephadex G-15 column (1.3 X 38 cm) which had been equilibrated with 0.02% NaNa solution to prevent bacterial growth. The column was eluted with 0.02% NaN3 solution at the rate of 1 ml/6 min. Approximately 0.35 ml was collected for each fraction and the radioactivity of each fraction was measured in 5 ml of Bray’s solution. The radioactivity of the polypeptide (in the void volume) and that of the oligopeptide (eluted before free amino acid) was measured. Further identification of the oligopeptidyl tRNA that were released was performed as described previously (6). RESULTS
Where indicated, erythromycin A was added. After incubating for 15 and 30 min at 3O”C, the amount of radioactive protein that was insoluble in hot (95’C) TCA in 80 ~1 of the mixture was measured by the filter disk method. For analysis of sedimentation behavior of polysomes, the mixture (0.35 ml) for protein synthesis was prepared essentially the same way as described above except that it contained 6 mM Mg acetate, 26 fig of RR factor free soluble enzymes, 1.37Azo, nm unit of polysomes without nascent peptides (16), and 230 pg of tRNA mixture containing 1.7 X lo5 cpm of ‘“C-valyl-, *4C-alanyl-, i4C-seryl-, and i4C-glycyl-tRNA (other aminoacyl-tRNAs were made with “C-amino acids). Where indicated, erythromycin A or sparsomycin was added. After incubating for 20 min at 30°C the mixture (300 ~1) was placed on 4.8 ml of a sucrose gradient (15-30% in 10 mM Tris-HCl (pH 7.4), 10 mM Mg acetate, 50 mM NH&l, and 1 mM DTT). The tubes were centrifuged for 55 min at 38,000 rpm in a SW 50.1 rotor. The sedimentation behavior of polysomes was analyzed with an ISCO densitometer and fractions were collected beginning at the top of the tube. The amount of radioactive protein that was insoluble in hot (95°C) TCA in 0.1 ml of each fraction was measured by the filter disk method. For analysis of the materials released from polysomes, the reaction mixture (0.35 ml) for protein synthesis was prepared essentially the same way as described in the preceding section except that it contained 510 fig of tRNA containing 2.1 X 10 cpm of i4C-valyl- and 19 “C-aminoacyl-tRNAs and 1.77Am nm units of puromycin-pretreated polysomes. After incubating for 30 min at 3O”C, the mixture was subjected to the sucrose density gradient centrifugation as described in the preceding section. Fractions were
Inhibitory effect of erythrmycin on protein synthesis by puromycin-treated polysomes. As shown in Table I, we first confirmed previously reported observations that erythromycin does not inhibit in vitro protein synthesis by naturally occurring polyribosomes (14). It appeared likely that the inability of erythromycin to inhibit protein synthesis in this system may be caused by steric hindrance exerted by the nascent peptidyl tRNA bound to ribosomes. If this is so, polyribosomes which have lost their peptidy1 group should be sensitive to this antibiotic, It should be pointed out that polyribosomes which have lost their nascent peptidyl group ordinarily undergo rapid conversion to monosomes in crude extract because the ribosome releasing factor (RR factor) releases such ribosomes from mRNA very rapidly (16, 17). Therefore, one would not be able to conduct in vitro protein biosynthesis by such polyribosomes. However, we have developed a method to remove only RR factor from a crude soluble fraction of E. coli (18), leaving all the other necessary components for protein synthesis. With this system, it is possible to study protein biosynthesis by polyribosomes which have been pretreated with puromycin to remove nascent polypeptides. These puromycintreated polyribosomes are fairly stable in the absence of RR factor and are able to produce polypeptides without going through the regular initiation steps. As shown in Table I, erythromycin exerts a very significant inhibitory action on the polyribosomes pretreated with puromycin. As low as lo-” M erythromycin A significantly inhibited incorporation of amino acids into polypeptides. These results snow that it is the presence of a long nascent polypeptide which inhibits the action of erythromycin.
OTAKA AND KAJI
848
Release of oligwpeptidyl tRNA from polysomes b erythromycin. In as much as erythromycin was found
to inhibit protein synthesis by polysomes, it was of interest to examine whether the mode of action proposed for the model system programmed by synthetic oligonucleotides holds for the naturally occurring polysomes. As shown in Table II, this is indeed the case. The amount of oligopeptidyl tRNA released from ribosomes in the presence of erythromycin was at least five times more than the control experiment. We conclude that, in the case of polyribosomes, erythromycin inhibits polypeptide synthesis by releasing oligopeptidyl tRNA from ribosomes. Release of ribawmes from mRNA after loss of oligopeptidyl tRNA. After erythromycin releases oli-
gopeptidyl tRNA from polysomes (Table II) the remaining ribosome-mRNA complex should not have the unesterified tRNA. This complex without tRNA is expected to be more unstable than polysomes with tRNA. Consistent with this expectation, erythromycin-treated polysomes are converted to monosomes rapidly even in the absence of RR factor (Fig. 1). In the control experiment, more polysomes remained (more than 50% of total ribosomes remained as polysomes in control while only 31% remained in the presence of erythromycin) even though the polysomes incorporated amino acids into polypeptides. This is in strong contrast to sparsomycin, a peptidyl transferase inhibitor, which inhibits amino acid incorporation without converting the polysomes to monosomes (Fig. Id).
TABLE II RELEASE OF OLIGOPEPTIDYL tRNA FROM POLYSOMES IN THE PRESENCE OF ERYTHROMYCIN A
Released peptidyl tRNA (cpm)
Antibiotic Erythromycin A (lo-5 M)
Total hot TCAinsoluble cpm
Polypeptide
Oligopeptide
9888
1111
175
2752
582
864
Note. The reaction mixture (0.35 ml) for synthesis of oligopeptide and polypeptides contained 20 mM Tris-HCl (pH 7.8), 50 ZUM NH&l, 8 mM Mg acetate, 1 mM DTT, 0.2 mM GTP, ‘7 mM phospboenolpyruvate, 8.9 ~g of pyruvate kinase, 280 pg E. co& soluble enzymes free of RR factor, 510 pg of tRNA containing 2.1 x lo5 cpm of ‘“C-valyl-tRNA, and 19 ‘*C-amino acyl-tRNA, and 0.80A2Banm unit of puromycinpretreated polysomes. After incubating for 30 min at 30% sucrose density gradient centrifugation was performed. The released peptidyl tRNA was isolated from the top of gradient. It was further separated into polypeptides and oligopeptide by Sephadex G-15 as described in the text.
0
5
IO
15 200 FRACTION
5
IO
15
20
NUMBER
FIG. 1. Sedimentation behavior of polysomes after incorporation of ‘%-amino acids in vitro. Effect of erythromycin A and sparsomycin. Puromycin pretreated polyribosomes were incubated with %-amino acids under the experimental condition described in the text. (a) No antibiotic and no incubation; (b) no antibiotic was added; (c) 10e4M erythromycin A was added; (d) 10e4 M sparsomycin was added. a, Hot TCA-insoluble radioactivity in each fraction; * . *, absorption at 254 nm. Sedimentation was from right to left.
DISCUSSION The data presented in this communication indicate that after removal of nascent peptidyl group from polyribosomes, one can observe the strong inhibitory effect of erythromycin on polysome-catalyzed amino acid incorporation. This is consistent with the observation of Pestka (14) that erythromycin can not bind to naturally occurring polyribosomes unless the peptidyl group is first removed. This is also in agreement with the observation that erythromycin binds to isolated ribosomes (21, 22). What is new in this report is that after removal of the peptidyl group, one can study the protein synthesis catalyzed by these pretreated polysomes. Ordinarily, upon removal of the peptidyl group, polyribosomes undergo rapid degradation into monosomes because of the presence of RR factor (16-20, 23). In the absence of RR factor, the puromycin-treated polysomes retain their structure and catalyze protein synthesis without the normal initiation steps. It is in this system, that we were able to demonstrate the significant inhibitory effect of erythromycin on polysome-catalyzed protein synthesis. Since erythromycin is shown to inhibit the polyribosome system in vitro, one can extend the present knowledge of the mechanism of erythromycin action into the in viva action. We postulate that erythromycin, in growing bacteria, binds to 50 S ribosome subunits (24, 25) of only polysomes with oligopeptidyl tRNA or no peptidyl tRNA. Once the nascent oligopeptides reach a certain size, eryth-
ERYTHROMYCIN
ACTION ON POLYRIBOSOME-CATALYZED
romycin can not stop further elongation of that chain and protein synthesis goes on until that ribosome completes translation of one cistron. In a sense, therefore, erythromycin acts as if it is an initiation inhibitor because it only binds to ribosomes with either no peptidyl tRNA or short peptidyl tRNA. This is consistent with the evidence, put forward by Tai et al. (ll), which states that erythromycin acts during the steps which closely follow the initiation step. The mode of action of erythromycin on polysomes appears to be similar to that in the model system programmed by synthetic polynucleotides. It releases oligopeptidyl tRNA from ribosomes. In a sense, erythromycin acts like puromycin which causes premature release of nascent polypeptides (26, 27). Erythromycin is different from puromycin in two important aspects, however: Erythromycin releases oligopeptidyl tRNA while puromycin releases the peptidyl group as peptidyl puromycin; puromycin releases all peptidyl groups regardless of their size while erythromycin releases only the oligopeptidyl group. This mode of action of erythromycin is consistent with the reported observation that erythromycin stimulates accumulation of oligopeptidyl tRNA in &JO in the absence of peptidyl tRNA hydrolase (28, 29). Since peptidyl tRNA is lethal to E. coli, erythromycin enhances the lethal effect of high temperature in the mutant having temperature-sensitive peptidyl tRNA hydrolase. These considerations render further support to the unified hypothesis (6) that erythromycin binds to the ribosomal site near the peptidyl transferase, resulting in inhibition of translocation (3, 4), and peptide bond formation (1, 2), while it stimulates the release of oligopeptidyl tRNA (6). Elucidation of the exact site of binding of this antibiotic to ribosomes (30, 31) would contribute toward further understanding of the mechanisms through which ribosomes carry out peptide bond formation and translocation. ACKNOWLEDGMENTS This work was supported by USPHS Grant GM 12053. The authors appreciate the help of Steven Vaupel in the preparation of this manuscript. REFERENCES 1. MAO, J. C.-H., AND ROBISHAW,E. E. (1972) Biochemistry
11, 4864-4872.
2. MAO, J.C.-H., AND ROBISHAW,E. E. (1971) Biochemistry
10, 2054-2061.
3. TANAKA, S., OTAKA, T., AND KAJI, A. (1973) Biochim.
Biophys. Acta 331,128-140.
4. IGARASHI,K., ISHITSUKA,H., AND KAJI, A. (1969) Biochem
Biophys. Res. Commun. 37,499-504.
5. CUNDLIFFE,E., AND MCQUILLEN,K. (1967) J. MoL BioL 30, 137-146.
PROTEIN BIOSYNTHESIS
849
6. OTAKA, T., AND KAJI, A. (1975) Proc. Nat. Acad Sci. USA 72,2649-2652.
7. MAJER, J. (1981) Antimicrob.
Agents Chemother.
19, 628-633.
8. FUJISAWA,Y., AND WEISBLUM,B. (1981) J. Bacteriol
146, 621.
9. HIROCHIKA, H. (1980) Mol. Gen. Genet. 179, 581588. 10. HORINOUCHI,S., AND WEISBLUM,B. (1980) Proc. Nat. Acad Sci. USA 77, 7079.
11. TAI, P. C., WALLACE,B. J., ANDDAVIS, B. D. (1974) Biochemistry 13,4653-4659. 12. MAO, J.C.-H., AND PUTERMAN,M. (1968) J. Bacteriol. W&1111-1117.
13. OLEINICK, N. L., AND CORCORAN,J. W. (1970) International Congress on Chemotherapy, 6th, Tokyo (Proc. I; pp. 202-ZOS),University Park, Baltimore, Md. 14. PESTKA, S. (1974) Antimicrob. Agents Chemother. 5, 255. 15. PESTKA, S. (1972) J. BioL Chem. 247.4669-467s. 16. HIRASHIMA, A., ANDKAJI, A. (1973) J. BioL Chem. 248, 7580-7587.
17. HIRASHIMA, A., ANDKAJI, A. (1972) Biochemistry 11, 4037-4044.
18. OGAWA,K., ANDKAJI, A. (1975) Biochim. Biophys. Acta 402, 288-296.
19. RYOJI, M., KARPEN, J. W., AND KAJI, A. (1981) J. BioL Chem. 256,5798.
20. RYOJI,M., BERLAND,R., ANDKAJI, A. (1981) Proc. Nat. Acad. Sci. USA 78, 5973.
21. TANAKA, K., TERAOKA, H., NAGIRA, T., AND TAMAKI, M. (1966) Biochim. Biophys. Acta 123, 435-437. 22. PESTKA,S. (1974) Antimicrob. Agents Chemother. 6, 474-478. 23. KUNG, H., TREADWELL,B. V., SPEARS,C., TAI, P, AND WEISSBACH,H. (1977) Proc. Natl. Acad Sci. USA 74,3217-3221. 24. WILHELM, J. M., ANDCORCORAN, J. W. (1967) Biochemistry 6, 2578.
25. FERNANDEZ-MUNOZ,R., MONRO,R. E., TORRES, PINEDO, R., AND VAZQUEZ,D. (1971) Eur. J. Biochem. 23, 185.
26. ALLEN, D. W., ANDZAMECNIK,P. C. (1962)B&him. Biophys. Acta 55, 865.
27. YARMOLINSKY,M., AND DE LA HABA (1959) Proc. Nat. Acad. Sci USA 45.1721-1729.
28. MENNINGER,J. R. (1976) J. BioL Chem. 251,33923398.
29. MENNINGER,J. R. (1979) J BacterioL
137, 694-
696.
30. LANGLOIS,R., LEE, C. C., CANTOR,C. R., VINCE, R., ANDPESTKA,S. (1976) .I MoL BioL 106,297313. 31. LANGLOIS, R., CANTOR, C. R., VINCE, R., AND PESTKA, S. (1977) Biochemistry 16,2349-2356.