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[14C]Erythromycin-ribosome complex formation and non-enzymatic binding of aminoacyl-transfer RNA to ribosome-messenger RNA complex
PRELIMINARY NOTES
435
BBA 91121
[t4C]Erythromycin-ribosome complex formation and non-enzymotic bindin 9 of aminoocyl-tronsfer RNA to ribosome-messenger RNA complex Erythromycin is known specifically to inhibit protein biosynthesis in susceptible bacteria 1-4. TAUBMAN et al. 5 showed that erythromycin blocks the protein biosynthetic pathway beyond the step of esterification of transfer RNA (tRNA). Recent studies in this laboratory have strongly suggested that the characteristic alteration of the ability of ribosomes in polyadenylic acid (poly A) directed polylysine synthesis was induced by the binding of erythromycin to ribosomes 6. NIRENBERG AND LEDER7 originated a simple filtration method for measuring the amount of [14C]aminoacyl-tRNA bound on a ribosome-messenger RNA (mRNA) complex taking advantage of the fact that ribosomes can be retained on Millipore
TABLE I THE BINDING
OF [ 1 4 C ] E R Y T H R O M Y C I N TO R I B O S O M E S
The complete E. coli system contained, in a final vol. of 5° #1; 5.43 A260 rn~ units of E. coli ribosomes (which contained i 7 6 # g of protein); [liC]erythromycin (8500 counts/rain); 5 #moles Trisacetate (pH 7.2); I # m o l e magnesium acetate; 2.5#moles KC1. The complete system for B. subtilis contained 11. 4 A260 ray units of B. subtilis ribosomes in place of the E. coli ribosomes, with other components as for the E. coli system. After incubation at 24 ° for 20 min, the reaction mixture was treated with 3 ml of pre-chilled buffer containing o.i M Tris-acetate (pH 7.2), 0.02 M magnesium acetate and 0.05 M KC1. The diluted reaction mixture was gently filtered on a Millipore filter (HA 0.45 #, 23 mm in diameter). The filter was washed with 3 ml of the same buffer (cold) 3 times, dried, and the radioactivity was counted using a Nuclear Chicago liquid-scintillation counter and toluene-PPO-l?OPOP phosphor solution. The background value (complete system minus ribosomes, 65 counts/min) was subtracted from the readings. The ribosome preparations used were obtained without deoxyribonuclease (EC 3.1.4.5) t r e a t m e n t and preincubation.
Expt. No.
Conditions
Radioactivity retained on filter (counts]rain)
Complete system (E. coli) with twice the amount of ribosomes Complete system (E. coli) plus non-labelled erythromycin A (o.2 m#mole) plus non-labelled erythromycin A (0. 4 m#mole) plus non-labelled erythromycin A (I m#mole) plus non-labelled erythromycin A (5 m/zmoles) Complete system (E. coli) plus tetracycline (2. 5 re#moles) plus tetracycline (lO m#moles) plus chloramphenicol (2. 5 m#moles) plus chloramphenicol (io re#moles) Complete system (B. subtilis) with one-half the amount of ribosomes with one-fourth the amount of ribosomes with one-eighth the amount of ribosomes
1171 2091 1196 803 541 377 lO3 lO16 lO52 987 lOO2 lO98 692 439 195 117
Abbreviations: tRNA, transfer RNA; poly A, polyadenylic acid; mRNA, messenger RNA; poly U, polyuridylic acid; POPOP, 1,4-bis(-5-phenyloxazolyl-2)-benzene; PPO, 2,5-diphenyl oxazole.
Biochim. Biophys. Acta, 123 (1966) 435-437
PRELIMINARY NOTES
436
filters. In the present investigation, this simple filtration method was found to be applicable for measuring the binding of [14Clerythromycin to ribosomes. It was also revealed that the binding of erythromycin to ribosomes dose not interfere with the non-enzymatic binding of [14Qaminoacyl-tRNA to the ribosome-mRNA complex. [l*CJErythromycin was obtained by short-term incubation of washed mycelium of Streptomyces erythreus with ~I-14Clpropionate (New England Nuclear Corp., 18. 4 mC/mmole) according to KANEDA et al. s. The paper chromatographic analysis 9 showed that approx. 78 % of the total radioactivity of this preparation was due to the erythromycin A and 17 % and 5 % of that were due to erythromycin C and B, respectively. Antimicrobial assay employed Bacillus subtilis (PCI-219) showed that 17 ooo counts/min of the preparation corresponded to approx. 0.43/*g of erythromycin A. Ribosomes and I o 5 o o o × g supernatant fraction (S-Ioo fraction) were obtained from Escherichia coli (I{-I2) and B. subtilis (PCI-219) as described by NIRENBERG AND MATTHAE110. E. coli ribosomal RNA was prepared from the ribosome preparation by phenol extraction without addition of sodium dodecyl sulfate 1°. ~14CILysyl-tRNA and [14C~phenylalanyl-tRNA were prepared according to the method of MOLDAVE11. As shown in Table I, [laC]erythromycin-ribosome complex was retained on Millipore filters (HA o.45#). No significant influence was observed when tetracycline and chloramphenicol were added to the incubation mixture. The addition of
TABLE l i T H E E F F E C T OF E R Y T H R O M Y C I N
ON TIlE
NON-ENZYMATIC BINDING OF AMINOACYL-tRNA TO
RIBOSOMES
The complete system for studying the binding of [14C]lysyl-tRNA to poly A -ribosomes contained the following components in a final vol. of 5°/zl: [14C]lysyl-tRNA (4200 counts/min, 17. 5/~/~moles) ; IO/zg poly A; 4.o3 A 26o m# units E. coli ribosomes (preincubated, well washedT); 5° m/zmoles non-labelled lysine; 5/zmoles Tris-acetate (pH 7.2); i / , m o l e magnesium acetate; 2.5/zmoles KC1. In the p*C~phenylalanyl-tlRNA-ribosome-polyU system, io/zg of poly U, [14C~phenylalanylt R N A (305° counts/rain 21.6 #,#*moles), and 5 ° m/zmoles of non-labelled phenylalanine were used in place of poly A, ?4C~lysyl tRNA, and non-labelled lysine. After the incubation at 24 ° for 20 rain, the reaction mixture was treated as described in the legend to Table I. Expt. No.
Conditions
Radioactivity retained on filter (counts~rain)
Complete system (poly A directed) minus poly A minus ribosomes minus poly A, plus poly U ( i o p g ) minus poly A and ribosomes plus [14C]erythromycin (17 ooocounts/min), minus ?4C]lysyl t R N A plus non-labelled erythromycin A (0.68 m/~mole) plus P4Clerythromycin (17 ooo counts/min) Complete system (poly U directed) minus poly U, plus poly A (io/~g) minus ribosomes plus [14C]erythromycin (17 ooo counts/min), minus [14C]phenylalanyl t R N A plus non-labelled erythromycin A (0. 5 m/zmole) plus [a*C]erythromycin
1352 54 ° 192 352 116
Biochim. Biophys. Mcta, 123 (1966) 435-437
980 168o 2665 1486 257 84 986 1538 2509
PREI.IMINARY NOTES
437
poly A, poly U, and E. coli ribosomal RNA had no meaningful effect on this complex formation. The specific activity of the complex was, however, decreased with the addition of increasing amounts of non-labelled erythromycin A to the incubation mixture. If it is assumed that the same amount of erythromycin was bound to the quantity of ribosomes used throughout the range of erythromycin concentrations and that no significant amount of erythromycin was liberated during the process of washing, thcn the specific activity of the original preparation of ~14CJerythromycin could be estimated to be 17 ooo counts/rain per o.66tlg as erythromycin A. This value was in fair agreement with the value obtained from the biological assay. By the use of this specific activity value, the amount of erythromycin bound to tile E. coli ribosome preparation used could further be estimated to be o.o46t~g of erythromycin A per 5.4 A,,6o,,,~, units. Similar results were obtained by column chromatographic separation on Sephadex G-5o of the [l*C~erythromycin-ribosome complex and the details of this study will be published elsewhere. These results seemed to be in harmony with the fact that the poly A directed [a4C)lysine incorporation into tungstate--trichloroacetic acid-insoluble materials was almost completely inhibited by the addition of o. 7 ¢tg of erythromycin A to 25oI~1 of the reaction mixture containing 25 A260m, units of E. coli ribosomes. The ia4Cjerythromycin--ribosome complex was also formed by the ribosomes obtained from B. subtilis (PCI-219). As shown in Table II, the non-enzymatic combination of [.~4C~lysyl tRNA with ribosome-poly A complex and that of [a4C]phenylalanyl tRNA with ribosomepoly U complex were not influenced by the binding of erythromycin to the ribosomes. We wish to thank Drs. T. KIMURA, K. MATSUMOTOAND M. MAYAMAfor their kind help in the preparation of ~14C]erythromycin.
Shionogi Research Laboratory, Shionogi and Co. Ltd., Fukushima-ku, Osaka (Japan)
KENTARO TANAKA HIROSHI TERAOKA TOMOYUKI NAGIRA MIKIO TAMAKI
1 P. BENIGNO, A. PORRO AND L. CIMA, Atti Accad. Naz. Lincei, Rend. Classe Sci. Fis. Mat. Nat., 16 (1954) 773. 2 H. NAKAGAWA, Osaka Daigaku Igaku Zasshi, 11 (I959) 345 I. 3 T. D. BROCK AND M. L. BROCK, Biochim. Biophys. Acta, 33 (1959) 274. 4 A. 1). ~VOLFE AND F. E. HAHN, Science, I43 (1904) 1445. 5 S. B. TAUBMAN, A. G. SO, l". E. YOUNG, (;. W. DAVIE AND J. W. CORCORAN, in J. C. SYLVESTER, Antimicrobial Agents and Chemotherapy, Am. Soc. Microbiol., Ann Arbor, Mich. 1964, p. 395. 6 i{. TANAKA AND tt. TEKAOKA, Biochim. Biophys. Acta, I14 (1966) 204. 7 M. NIRENBERG AND P. LEOER, Science, 145 (I964) 1399. 8 T. KANEDA, J. C. BUTTE, S. B. TAUBMAN AND J. w . CORCORAN, J. Biol. Chem., 237 (I962) 322 9 P. P. I-tUNG, C. I,. MARKS AND ['. L. TARDREW, J. Biol. Chem., 24o (I965) I322. IO .'~I. W. NIRENBERG AND J. H. MATTHAEI, Proc. Natl. Acad. Sci. U.S., 47 (I961) r588. i I K. M'OLDAVE, in S. P. COLOWICK AND •. O. KAPLAN, Methods in Enzymology, Vol. 6, Academic l~ress, New York, 1964, p. 757.
Received April 26th, 1966 Biochim. Biophys. Acta, 12 3 (I966) 435-437
435
BBA 91121
[t4C]Erythromycin-ribosome complex formation and non-enzymotic bindin 9 of aminoocyl-tronsfer RNA to ribosome-messenger RNA complex Erythromycin is known specifically to inhibit protein biosynthesis in susceptible bacteria 1-4. TAUBMAN et al. 5 showed that erythromycin blocks the protein biosynthetic pathway beyond the step of esterification of transfer RNA (tRNA). Recent studies in this laboratory have strongly suggested that the characteristic alteration of the ability of ribosomes in polyadenylic acid (poly A) directed polylysine synthesis was induced by the binding of erythromycin to ribosomes 6. NIRENBERG AND LEDER7 originated a simple filtration method for measuring the amount of [14C]aminoacyl-tRNA bound on a ribosome-messenger RNA (mRNA) complex taking advantage of the fact that ribosomes can be retained on Millipore
TABLE I THE BINDING
OF [ 1 4 C ] E R Y T H R O M Y C I N TO R I B O S O M E S
The complete E. coli system contained, in a final vol. of 5° #1; 5.43 A260 rn~ units of E. coli ribosomes (which contained i 7 6 # g of protein); [liC]erythromycin (8500 counts/rain); 5 #moles Trisacetate (pH 7.2); I # m o l e magnesium acetate; 2.5#moles KC1. The complete system for B. subtilis contained 11. 4 A260 ray units of B. subtilis ribosomes in place of the E. coli ribosomes, with other components as for the E. coli system. After incubation at 24 ° for 20 min, the reaction mixture was treated with 3 ml of pre-chilled buffer containing o.i M Tris-acetate (pH 7.2), 0.02 M magnesium acetate and 0.05 M KC1. The diluted reaction mixture was gently filtered on a Millipore filter (HA 0.45 #, 23 mm in diameter). The filter was washed with 3 ml of the same buffer (cold) 3 times, dried, and the radioactivity was counted using a Nuclear Chicago liquid-scintillation counter and toluene-PPO-l?OPOP phosphor solution. The background value (complete system minus ribosomes, 65 counts/min) was subtracted from the readings. The ribosome preparations used were obtained without deoxyribonuclease (EC 3.1.4.5) t r e a t m e n t and preincubation.
Expt. No.
Conditions
Radioactivity retained on filter (counts]rain)
Complete system (E. coli) with twice the amount of ribosomes Complete system (E. coli) plus non-labelled erythromycin A (o.2 m#mole) plus non-labelled erythromycin A (0. 4 m#mole) plus non-labelled erythromycin A (I m#mole) plus non-labelled erythromycin A (5 m/zmoles) Complete system (E. coli) plus tetracycline (2. 5 re#moles) plus tetracycline (lO m#moles) plus chloramphenicol (2. 5 m#moles) plus chloramphenicol (io re#moles) Complete system (B. subtilis) with one-half the amount of ribosomes with one-fourth the amount of ribosomes with one-eighth the amount of ribosomes
1171 2091 1196 803 541 377 lO3 lO16 lO52 987 lOO2 lO98 692 439 195 117
Abbreviations: tRNA, transfer RNA; poly A, polyadenylic acid; mRNA, messenger RNA; poly U, polyuridylic acid; POPOP, 1,4-bis(-5-phenyloxazolyl-2)-benzene; PPO, 2,5-diphenyl oxazole.
Biochim. Biophys. Acta, 123 (1966) 435-437
PRELIMINARY NOTES
436
filters. In the present investigation, this simple filtration method was found to be applicable for measuring the binding of [14Clerythromycin to ribosomes. It was also revealed that the binding of erythromycin to ribosomes dose not interfere with the non-enzymatic binding of [14Qaminoacyl-tRNA to the ribosome-mRNA complex. [l*CJErythromycin was obtained by short-term incubation of washed mycelium of Streptomyces erythreus with ~I-14Clpropionate (New England Nuclear Corp., 18. 4 mC/mmole) according to KANEDA et al. s. The paper chromatographic analysis 9 showed that approx. 78 % of the total radioactivity of this preparation was due to the erythromycin A and 17 % and 5 % of that were due to erythromycin C and B, respectively. Antimicrobial assay employed Bacillus subtilis (PCI-219) showed that 17 ooo counts/min of the preparation corresponded to approx. 0.43/*g of erythromycin A. Ribosomes and I o 5 o o o × g supernatant fraction (S-Ioo fraction) were obtained from Escherichia coli (I{-I2) and B. subtilis (PCI-219) as described by NIRENBERG AND MATTHAE110. E. coli ribosomal RNA was prepared from the ribosome preparation by phenol extraction without addition of sodium dodecyl sulfate 1°. ~14CILysyl-tRNA and [14C~phenylalanyl-tRNA were prepared according to the method of MOLDAVE11. As shown in Table I, [laC]erythromycin-ribosome complex was retained on Millipore filters (HA o.45#). No significant influence was observed when tetracycline and chloramphenicol were added to the incubation mixture. The addition of
TABLE l i T H E E F F E C T OF E R Y T H R O M Y C I N
ON TIlE
NON-ENZYMATIC BINDING OF AMINOACYL-tRNA TO
RIBOSOMES
The complete system for studying the binding of [14C]lysyl-tRNA to poly A -ribosomes contained the following components in a final vol. of 5°/zl: [14C]lysyl-tRNA (4200 counts/min, 17. 5/~/~moles) ; IO/zg poly A; 4.o3 A 26o m# units E. coli ribosomes (preincubated, well washedT); 5° m/zmoles non-labelled lysine; 5/zmoles Tris-acetate (pH 7.2); i / , m o l e magnesium acetate; 2.5/zmoles KC1. In the p*C~phenylalanyl-tlRNA-ribosome-polyU system, io/zg of poly U, [14C~phenylalanylt R N A (305° counts/rain 21.6 #,#*moles), and 5 ° m/zmoles of non-labelled phenylalanine were used in place of poly A, ?4C~lysyl tRNA, and non-labelled lysine. After the incubation at 24 ° for 20 rain, the reaction mixture was treated as described in the legend to Table I. Expt. No.
Conditions
Radioactivity retained on filter (counts~rain)
Complete system (poly A directed) minus poly A minus ribosomes minus poly A, plus poly U ( i o p g ) minus poly A and ribosomes plus [14C]erythromycin (17 ooocounts/min), minus ?4C]lysyl t R N A plus non-labelled erythromycin A (0.68 m/~mole) plus P4Clerythromycin (17 ooo counts/min) Complete system (poly U directed) minus poly U, plus poly A (io/~g) minus ribosomes plus [14C]erythromycin (17 ooo counts/min), minus [14C]phenylalanyl t R N A plus non-labelled erythromycin A (0. 5 m/zmole) plus [a*C]erythromycin
1352 54 ° 192 352 116
Biochim. Biophys. Mcta, 123 (1966) 435-437
980 168o 2665 1486 257 84 986 1538 2509
PREI.IMINARY NOTES
437
poly A, poly U, and E. coli ribosomal RNA had no meaningful effect on this complex formation. The specific activity of the complex was, however, decreased with the addition of increasing amounts of non-labelled erythromycin A to the incubation mixture. If it is assumed that the same amount of erythromycin was bound to the quantity of ribosomes used throughout the range of erythromycin concentrations and that no significant amount of erythromycin was liberated during the process of washing, thcn the specific activity of the original preparation of ~14CJerythromycin could be estimated to be 17 ooo counts/rain per o.66tlg as erythromycin A. This value was in fair agreement with the value obtained from the biological assay. By the use of this specific activity value, the amount of erythromycin bound to tile E. coli ribosome preparation used could further be estimated to be o.o46t~g of erythromycin A per 5.4 A,,6o,,,~, units. Similar results were obtained by column chromatographic separation on Sephadex G-5o of the [l*C~erythromycin-ribosome complex and the details of this study will be published elsewhere. These results seemed to be in harmony with the fact that the poly A directed [a4C)lysine incorporation into tungstate--trichloroacetic acid-insoluble materials was almost completely inhibited by the addition of o. 7 ¢tg of erythromycin A to 25oI~1 of the reaction mixture containing 25 A260m, units of E. coli ribosomes. The ia4Cjerythromycin--ribosome complex was also formed by the ribosomes obtained from B. subtilis (PCI-219). As shown in Table II, the non-enzymatic combination of [.~4C~lysyl tRNA with ribosome-poly A complex and that of [a4C]phenylalanyl tRNA with ribosomepoly U complex were not influenced by the binding of erythromycin to the ribosomes. We wish to thank Drs. T. KIMURA, K. MATSUMOTOAND M. MAYAMAfor their kind help in the preparation of ~14C]erythromycin.
Shionogi Research Laboratory, Shionogi and Co. Ltd., Fukushima-ku, Osaka (Japan)
KENTARO TANAKA HIROSHI TERAOKA TOMOYUKI NAGIRA MIKIO TAMAKI
1 P. BENIGNO, A. PORRO AND L. CIMA, Atti Accad. Naz. Lincei, Rend. Classe Sci. Fis. Mat. Nat., 16 (1954) 773. 2 H. NAKAGAWA, Osaka Daigaku Igaku Zasshi, 11 (I959) 345 I. 3 T. D. BROCK AND M. L. BROCK, Biochim. Biophys. Acta, 33 (1959) 274. 4 A. 1). ~VOLFE AND F. E. HAHN, Science, I43 (1904) 1445. 5 S. B. TAUBMAN, A. G. SO, l". E. YOUNG, (;. W. DAVIE AND J. W. CORCORAN, in J. C. SYLVESTER, Antimicrobial Agents and Chemotherapy, Am. Soc. Microbiol., Ann Arbor, Mich. 1964, p. 395. 6 i{. TANAKA AND tt. TEKAOKA, Biochim. Biophys. Acta, I14 (1966) 204. 7 M. NIRENBERG AND P. LEOER, Science, 145 (I964) 1399. 8 T. KANEDA, J. C. BUTTE, S. B. TAUBMAN AND J. w . CORCORAN, J. Biol. Chem., 237 (I962) 322 9 P. P. I-tUNG, C. I,. MARKS AND ['. L. TARDREW, J. Biol. Chem., 24o (I965) I322. IO .'~I. W. NIRENBERG AND J. H. MATTHAEI, Proc. Natl. Acad. Sci. U.S., 47 (I961) r588. i I K. M'OLDAVE, in S. P. COLOWICK AND •. O. KAPLAN, Methods in Enzymology, Vol. 6, Academic l~ress, New York, 1964, p. 757.
Received April 26th, 1966 Biochim. Biophys. Acta, 12 3 (I966) 435-437