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Reaction of puromycin with N-acetylphenylalanyl-tRNA on ribosomes reassociated from escherichia coli ribosomal subunits
304
BIOCHIMICA ET BIOPHYSICA A('TA
BBA 9702.1
REACTION OF PUROMYCIN W I T H N-ACETYLPHENYLALANYL-tRNA ON RIBOSOMES REASSOCIATED FROM E S C H E R I C H I A COLI RIBOSOMAL SUBUNITS H I R O S H I T E R A O K A AND K E N T A R O TANAKA
Shionogi Research Laboratory, Shionogi and Co., Ltd., Fukushima-ku, Osaka (Japan) (Received J u n e i8th, 1971)
SUMMARY
N-Acetylphenylalanylpuromycin synthesis catalyzed by ribosomes reassociated from 5o-S and 3o-S subunits was studied. N-Acetylphenylalanylpuromycin was formed even at o ° by addition of puromycin to a mixture of 5o-S ribosomal subunits and a preparation of 3o-S ribosomal subunits, poly(U) and N-acetylphenylalanyl-tRNA previously mixed at o ° after preincubation under suitable conditions. To obtain maximal formation of N-acetylphenylalanylpuromycin by reassociated ribosomes, the 5o-S ribosomal subunits had to be preincubated at a sufficient concentration of NH4÷ (or K ÷) at a suitable temperature (about 37°), but a change in the concentration of Mg~+ showed no significant effect. The preincubation of 3o-S ribosomal subunits did not require a particular concentration of NH4 + (or K +) but a sufficient Mg2+ concentration was necessary. Using this technique we were able to study the effect of environmental conditions on the activity of respective ribosomal subunits in peptide synthesis. The 5o-S and 3o-S subunits preparations were obtained from ribosomes of parent Escherichia coli QI3 strain and from its mutant ribosomes which have an altered specific protein component in the 5o-S subunit and a high monovalent cation (K + or NH4+ ) dependency in the peptidyl transfer reaction. The hybrid ribosomes consisted of QI3 3o-S subunits and the mutant 5o-S subunits showed distinctly higher monovalent cation dependency than the hybrid ribosomes from QI 3 5o-S subunits and the 3o-S subunits of the mutant ribosomes, suggesting that alteration of 5o-S ribosomal subunits induces alteration of their peptidyl transferase activity.
INTRODUCTION
The puromycin reaction has been considered as a simple model of peptide bond formation. The reaction is catalyzed by peptidyl transferase, an enzyme which seems to be an integral part of the 5o-S subunit of Escheriehia eoli ribosomes 1,2. Furthermore, MISKIN et al. 3,4 have suggested that the activity of this enzyme depends upon the ribosome conformation which is reversibly altered with changes in the concentiation of K+ or N H , +. Abbreviations: Ac-Phe-tlRNA, N-acetylphenylalanyl-tRNA; Ac-Phe-puromycin, N-acetylphenylalanylpuromycin.
Biochim. Biophys. Acta, 247 (1971) 304-309
RIBOSOMAL PEPTIDYL TRANSFER REACTION
305
It has been shown that the reaction between Ac-Phe-tRNA and puromycin occurs readily on salt-washed 7o-S ribosomes in the presence of poly(U), Mg2+, and monovalent cation such as K + or NH4+ (refs. 5, 6). Using this system, we have previously found that ribosomes from the mutants of E. coli having an altered specific 5o-S ribosomal protein component (5o-8), have lower peptidyl transferase activity than those from parent QI3 cells at ordinary salt concentration 7. These ribosomes "less active" in peptidyl transferase activity have been shown to be convertible to the "active" state by increasing the environmental K + (or NH4+ ) concentration. To obtain further insight into the activity of ribosomes in the peptidyl transfer reaction, we describe here the Ieaction of puromycin with Ac-Phe-tRNA on ribosomes reconstituted from 3o-S and 5o-S ribosomal subunits. The results suggest that the function of 5o-S ribosomal subunits is affected by the concentration of N H , + more specifically than by that of Mg2+, and indicate that the chemical structure of the 50-8 ribosomal protein component is closely correlated with ribosomal peptidyl transferase activity.
MATERIALS AND METHODS
Ribosomes were prepared according to the method of NIRENBERG8 from E. coli QI3 and E. coli QE2oI (erythromycin-resistant mutant derived from QI3 as
described previouslyg). Ribosomal subunits were prepared by sucrose gradient centrifugation after dissociation by the addition of phosphate 1°, and were dialyzed against o.oi M Tris buffer (pH 7.4) containing o.oi M magnesium acetate. Ac-[a4C]Phe-tRNA was prepared by the method of LAPIDOT et al. 11. Puromycin • 2HCI was purchased from the Nutritional Biochemical Corp. and poly(U) was obtained from C. F. Boehringer and Sohn GmbH. [14C]Phenylalanine (specific radioactivity 455/~C//~mole) was obtained from Schwarz BioResealch Inc. The assay for the formation of Ac-E14C]Phe-puromycin was a modification of the method described by LEDER AND BURSZTYN12 for the assay of N-formylmethionylpuromycin. The reaction conditions are specified in the legends to the tables and figure. After incubation the reaction was stopped by the addition of 0.25 ml of 0. 4 IV[ sodium acetate (pH 5.5), then extracted with 1.5 ml of ethyl acetate with shaking. After centrifugation at 2000 rev./min for IO min, I ml of the ethyl acetate layer was transferred to a scintillation vial containing IO ml of BRAY'Sls solution and radioactivity was counted in a Beckman liquid scintillation counter.
RESULTS AND DISCUSSION
Although it was reported by MONRO1 that the reaction of puromycin with Nblocked aminoacyloligonucleotide fragment from tRNA takes place on 5o-S ribosomal subunits alone in the presence of alcohol, it is evident from Table I, that both ribosomal subunits are required for the formation of Ac-Phe-puromycin in this system. Table I also shows that, as expected from previous work employing 70-S ribosomes 14, Ac-Phe-puromycin was formed at o ° when the preincubation of ribosomal subunits was carried out in the presence of sufficient Mg 2+ and NH4+. Biochim. Biophys. Acta, 247 (1971) 3o4-3o9
306 TABLE
H. TERAOKA, K. TANAKA I
Ac-Phe-PUROMYClN
FORMATION DEPENDING
UPON BOTH RIBOSOMAL SUBUNITS
Expt. I: A m i x t u r e (Ioo #1) contained 4 °/2g of poly(U), 0.72 Ase 0 nm unit of Ac-[I*C~Phe-tRNA (12 ooo c o u n t s / m i n ) and indicated a m o u n t s of 3o-S s u b u n i t s in a s t a n d a r d buffer (5 ° mM T r i s HC1, p H 7.8, 16 mM m a g n e s i u m acetate, 0.2 M NH4C1 ). A suspension of 5o-S s u b u n i t s (ioo/~1) contained indicated a m o u n t s of 5o-S s u b u n i t s in the s t a n d a r d buffer. The m i x t u r e containing 3o-S s u b u n i t s and the suspension of 5o-S s u b u n i t s were p r e i n c u b a t e d at 37" for io nlin, cooled in ice for 5 rain, t h e n combined t o g e t h e r at o °. The p u r o m y c i n reaction (225/,1) was s t a r t e d b y the addition of o . I / * m o l e p u r o m y c i n (dissolved in the s t a n d a r d buffer) to the combined mixture, a n d the reaction t u b e was incubated at o ° for io min. The a s s a y for A c - P h e - p u r o m y c i n formation was carried o u t as described u n d e r MATERIALS AND M E T H O D S , Expt. 2: E x p e r i m e n t a l conditions were the same as described in E x p t . I, except t h a t after preincubation, the m i x t u r e containing 3o-S s u b u n i t s a n d the suspension of 5o-S were combined together w i t h o u t cooling in ice, kept at 37 ° for 3 rain, t h e n cooled to o °.
Expt.
3o-S subunits (A2~0 nm units)
5o-S subunits (A,6o nmUnits)
Ac-Phe-puromycin /ormed (counts/rain)
I
o 0.64 0.32 0.64
1.28 o 0.64 1.28
26 45 585 116o
2
0.64
1.28
1264
It is of interest to note that almost the same level of Ac-Phe-puromycin formation was observed whether the combination of 5o-S and 3o-S subunits of ribosomes was carried out at o ° or 37 ° (Table I, Expts. I and 2). This fact may indicate that association of the two ribosomal subunits takes place even at o °. To obtain further information on the effect of different preincubation conditions on the ability of each subunit to participate in Ac-Phe-puromycin formation, reactions were carried out in which the composition of the reaction mixture and the reaction temperature (o°) were kept constant, any differences in the amounts of Ac-Phe-puromycin formed thus being mainly due to differences in the preincubation conditions. TABLE II ~EEBCT OF TEMPERATURE
IN THE PREINCUBATION
STEP
E x p e r i m e n t a l conditions were the same as described in Table I, E x p t . I, except t h a t the m i x t u r e containing 3o-S s u b u n i t s and t h e suspension of 5o-S s u b u n i t s contained 0.66 A,~ 0 nm unit of 3o-S s u b u n i t s and I.O6 A ~60 nm units of 5o-S subunits, respectively, p r e i n c u b a t e d at the indicated temperatures,
Preineubation temperature Mixture containing 3o-S subunits
Suspension o[ 5o-S subunits
Ac-Phe-puromycin [ormed (counts~rain)
37 ° 37 ° o°
37 ° °Q 37 °
908 47 399
0°
0°
18
As shown in Table I1, preincubation of 5o-S subunits must be carried out at a suitable temperature (37 °) for the subsequent puromycin reaction. When the preBiochim. Biophys. Aeta, 247 (1971) 3o4-3o9
307
RIBOSOMAL PEPTIDYL TRANSFER REACTION
incubation was carried out at low temperature (o°), 5o-S subunits showed no significant activity even if other conditions, such as environmental ionic conditions, were sufficient. When a mixture containing 3o-S subunits, poly(U) and Ac-Phe-tRNA was preincubated at o °, however, significant but not maximum synthesis of AcPhe-puromycin was observed. This fact suggests that the 3o-S subunit preparation used in this study still retains the ability to bind Ac-Phe-tRNA at o °. As already reported 5,6,15, the Ac-Phe-puromycin forming activity of 7o-S ribosomes in the presence of poly(U) is strongly dependent upon the concentration of Mg ~+ and monovalent cations such as K + or NH4+, maximum activity being obtained with lO-2O mlV[ Mg 2. and o.16-o.2 M NH4+. When 5o-S subunits were preincubated in the medium depleted of K + or NH4+, no detectable formation of Ac-Phepuromycin was observed at o ° even in the presence of sufficient NH4+ in the reaction mixture (Table III). Considered together with the results listed in Table II, the pep-
TABLE
Ill
E F F E C T OF N H 4 +
CONCENTRATION
IN T H E P E E I N C U B A T I O N
MEDIUM
P r e i n c u b a t i o n conditions were the same as described in Table I, E x p t . I, except t h a t the m i x t u r e containing 3o-S s u b u n i t s and the suspension of 5o-S s u b u n i t s contained 0.53 A,e 0 nm u n i t of 3o-S s u b u n i t s and 1.o2 A2e0 nm units of 5o-S subunits, respectively, p r e i n c u b a t e d at 37 ° in a m e d i u m containing 16 mM Mg 2+ and indicated concentrations of NH~ +. Prior to the p u r o m y c i n reaction, the ionic concentration of each combined m i x t u r e was adjusted to the same value (o.2 M NH4+, 16 mM Mg2+), t h e n o . I / * m o l e p u r o m y c i n (dissolved in the s t a n d a r d buffer) was added to m a k e a final v o l u m e of 225/21. O t h e r conditions were the same as described in Table I, E x p t . I.
NHa + concentration (M) I n the mixture containing 3o-S subunits
I n the suspension o / 5 o - S subunits
0.2 0.2 o o
0.2 o 0.2 o
A c-Phe-puromycin /ormed (counts~rain)
778 26 75 ° 17
tidyl transferase function of 5o-S ribosomal subunits appears to be activated during the preincubation step in the presence of sufficient NH4 +, the activation rate being largely dependent upon the preincubation temperature. These results are consistent with the suggestion of MISKIN et al. 3,4 that the fragment reaction catalyzed b y ribosomes is largely dependent upon the conformation of ribosomes, which is reversible depending on the K + (or NH4+ ) concentration. The preincubation of 5o-S ribosomal subunits at very low Mg2+ concentration caused no detectable decrease in Ac-Phepuromycin formation (Table IV). This observation strongly supports our previous finding that the affinity of ribosomes (5o-S subunits) for erythromycin is dependent on the environmental concentration of K + or NH4 + but not on that of Mg2+ (see refs. 16, I7) , this being in marked contrast to the cation dependency of preincubation mixtures containing 3o-S subunits. These facts suggest that the function of 5o-S ribosomal subunits in the process of peptide bond formation in ribosomes is, in contrast to that of 3o-S subunits, affected b y the concentration of monovalent cations such as K + or NH~ + more specifically than it is by the concentration of Mg2+. Biochim. Biophys. Acta, 247 (1971) 3o4-3o9
308
H. TERAOKA, K. TANAKA
TABLE IV E F F E C T OF ~V[g2+ CONCENTRATION IN THE PRI~INCUBATION MEDIUM
P r e i n c u b a t i o n conditions were the same as described in Table I, E x p t . i, except t h a t the m i x t u r e containing 3o-S s u b u n i t s and the suspension of 5o-S s u b u n i t s contained o.66 A~60 nm unit of 3o-S and 1.o6 A,e 0 nm units of 5o-S subunits, respectively, p r e i n c u b a t e d at 37 ° in a m e d i u m containing o.2 1V[ NH4+ and indicated concentrations of Mg 2+. O t h e r conditions were the same as described in Table I I I .
Mg 2+ concentration (raM) In the mixture containing 3o-S subunits
In the suspension o/5o-S subunits
16.o 16.o 2.0 2.0
16.o 2.5 16.o 2.5
A c- Phe-puromycin [orrned (counts/min)
846 895 285 254
We have previously shown that the ribosomes from the erythromycin resistant mutants of E. coli, having an altered specific 5o-S ribosomal protein component (5o-8), have distinctly lowered activity in Ac-Phe-puromycin formation, although their ability to bind Ae-Phe-tRNA is more or less unaltered ~. The Ac-Phe-puromycin forming activity of these ribosomes increased very sharply with increase in environmental K + or NH4 + concentration, and reached the same maximum level as that observed for parent QI3 ribosomes at sufficient concentration of these monovalent cations. From these facts, we suggested that a structural change in a protein component influences the conformation of 5o-S ribosomal subunit and induces consequent alteration of the peptidyl transferase activity. To obtain further insight into this, hybrid 7o-S ribosomes were obtained from 3o-S and 5o-S ribosomal subunits prepared from QI3 and its mutant (QE2oI), and the ability of such hybrid ribosomes to function in Ac-Phe-puromycin formation was then tested. As shown by the data in Fig. I, hybrid ribosomes containing QE2oI 5o-S subunits have much lower activity than hybrid ribosomes containing QI 3 5o-S subunits. E_ 1~ 100C
E
~
c_. 5oc
....
./. KCI (raM)
NHaC[ (mM)
Fig.
I. A c - P h e - p u r o m y c i n forming activity of h y b r i d ribosomes obtained from ribosomal subu n i t s of Q I 3 and its m u t a n t . The reaction m i x t u r e contained the following c o m p o n e n t s in a final v o l u m e of 125 #1; 5 ° mlV~ Tris-tiC1 (pH 7.8), 16 mM m a g n e s i u m acetate, o.42 A260 nm unit of Ac-E14C]Phe-ttZNA (93oo counts/rain), 4 ° / z g of poly(U), 0. 4 A2~0 nm unit of 3o-S subunits, o.8 M260 nm u n i t of 5o-S subunits, o. 4 mM puromycin, and a specified concentration of K + or NHa+. I n c u b a t i o n was carried o u t at 37 ° for io min. The assay for A c - P h e - p u r o m y c i n f o r m a t i o n was carried o u t as described u n d e r MATERIALS AND METHODS. O - - O , Q E 2 o I 3o-S s u b u n i t s and Q I 3 5o-S subunits. O - - - O , QI3 3o-S s u b u n i t s and Q E 2 o I 5o-S subunits.
Biochim. Biophys. Acta, 247 (1971) 304-309
309
RIBOSOMAL PEPTIDYL TRANSFER REACTION
R e c e n t l y i t w a s f o u n d t h a t t h e c h a r a c t e r of t h e a l t e r e d 50-8 p r o t e i n c o m p o n e n t is a l w a y s c o - t r a n s d u c e d w i t h t h e e r y t h r o m y c i n r e s i s t a n c e f r o m d o n o r t o recip i e n t b a c t e r i a b y P l k c p h a g e TM. I t w a s f u r t h e r s h o w n t h a t a l t e r a t i o n of 50-8 p r o t e i n c o m p o n e n t i n r i b o s o m e s f r o m t h e s e t r a n s d u c t a n t s is a l w a y s a c c o m p a n i e d b y r e d u c e d r i b o s o m a l p e p t i d y l t r a n s f e r a s e a c t i v i t y TM. T o g e t h e r w i t h t h e r e s u l t s i n Fig. I, t h e s e r e s u l t s g i v e s t r o n g e v i d e n c e t h a t t h e c h e m i c a l s t r u c t u r e of t h e 50-8 r i b o s o m a l p r o t e i n c o m p o p e n t is c l o s e l y c o r r e l a t e d w i t h t h e p e p t i d e s y n t h e s i z i n g a b i l i t y of r i b o s o m e s .
ACKNOWLEDGMENT W e t h a n k Mr. M. T a m a k i f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e .
t~EFERENCES i 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 I8 19
R. E. MONRO, J. ~V[ol. Biol., 26 (1967) 147. I3. E. H. ~-~[ADEN, R. 1~. TRA.UT AND R. E. ~tV[ONRO,J. Mol. Biol., 35 (1968) 333. R. MISKIN, A. ZAMIR AND D. ELSON, Biochem. Biophys. Res. Commun., 33 (1968) 551. R. MISKIN, A. ZAMIR AND D. ELSON, J. Mol. Biol., 54 (197o) 355. J. LUCA.s-LENARD A.ND F. LIPMANN, Proc. Natl. Acad. Sci. U.S., 57 (1967) Io5o. H. WEISSBACH, 13. REDFIELD AND N. BROT, Arch. Biochem. Biophys., I27 (1968) 705. H. TERAOKA, ~Y[. TA.MAKI AND K. TANAKA, Biochem. Biophys. Res. Commun., 38 (197 o) 328. 1V[.W. N'IRENBERG, in S. P. COLOWlCK AND N. O. HA.PLAN, Methods in Enzymology, Vol. 6, Academic Press, New York, 1963, p. I7. E. OTA.KA, H. TERAOKA, M. TAMAKI, K. TANAKA.AND S. OSAWA., J. Mol. Biol., 48 (197 o) 499. A. TlSSI~RES, J. D. WATSON, D. SCHLESSINGER AND B. R. HOLLINGWORTH, J. Mol. Biol., i (1959) 221. Y. LAPIDOT, I'4. DE GROOT, I. FRY-SHAFRIR, Biochim. Biophys. Acta, 145 (I967) 292. P. LEI)ER A.ND H. BURSZTYN, Biochem. Biophys. Res. Commun., 25 (1966) 233. A. G. ]3RAY, Anal. Biochem., I (196o) 279. J. M. RAVEL, R. L. SHOREY AND W. SHIVE, Biochemistry, 9 (197 o) 5 °28. I-I. TERAOKA AND I~. TANAKA, Biochim. Biophys. Acta, 232 (1971) 5o9 . H. TERAOKA, J. Mol. Biol., 48 (197 o) 511. H. TERA.OKA, J. Antibiot., 24 (1971) 302. R. TAKA.TA, S. OSAWA, K. TANAKA, H. TERAOKA.AND M. TAMAKI, Mol. Gen. Genet., lO9 (197 o) 123. K. TANAKA, H. TERA.OKA, ~-~. TA.MAKI, R. TAKATA AND S. OSAWA, submitted to Mol. Gren. Genet.
Biochim. Biophys. Acta, 247 CI97I) 304-309
BIOCHIMICA ET BIOPHYSICA A('TA
BBA 9702.1
REACTION OF PUROMYCIN W I T H N-ACETYLPHENYLALANYL-tRNA ON RIBOSOMES REASSOCIATED FROM E S C H E R I C H I A COLI RIBOSOMAL SUBUNITS H I R O S H I T E R A O K A AND K E N T A R O TANAKA
Shionogi Research Laboratory, Shionogi and Co., Ltd., Fukushima-ku, Osaka (Japan) (Received J u n e i8th, 1971)
SUMMARY
N-Acetylphenylalanylpuromycin synthesis catalyzed by ribosomes reassociated from 5o-S and 3o-S subunits was studied. N-Acetylphenylalanylpuromycin was formed even at o ° by addition of puromycin to a mixture of 5o-S ribosomal subunits and a preparation of 3o-S ribosomal subunits, poly(U) and N-acetylphenylalanyl-tRNA previously mixed at o ° after preincubation under suitable conditions. To obtain maximal formation of N-acetylphenylalanylpuromycin by reassociated ribosomes, the 5o-S ribosomal subunits had to be preincubated at a sufficient concentration of NH4÷ (or K ÷) at a suitable temperature (about 37°), but a change in the concentration of Mg~+ showed no significant effect. The preincubation of 3o-S ribosomal subunits did not require a particular concentration of NH4 + (or K +) but a sufficient Mg2+ concentration was necessary. Using this technique we were able to study the effect of environmental conditions on the activity of respective ribosomal subunits in peptide synthesis. The 5o-S and 3o-S subunits preparations were obtained from ribosomes of parent Escherichia coli QI3 strain and from its mutant ribosomes which have an altered specific protein component in the 5o-S subunit and a high monovalent cation (K + or NH4+ ) dependency in the peptidyl transfer reaction. The hybrid ribosomes consisted of QI3 3o-S subunits and the mutant 5o-S subunits showed distinctly higher monovalent cation dependency than the hybrid ribosomes from QI 3 5o-S subunits and the 3o-S subunits of the mutant ribosomes, suggesting that alteration of 5o-S ribosomal subunits induces alteration of their peptidyl transferase activity.
INTRODUCTION
The puromycin reaction has been considered as a simple model of peptide bond formation. The reaction is catalyzed by peptidyl transferase, an enzyme which seems to be an integral part of the 5o-S subunit of Escheriehia eoli ribosomes 1,2. Furthermore, MISKIN et al. 3,4 have suggested that the activity of this enzyme depends upon the ribosome conformation which is reversibly altered with changes in the concentiation of K+ or N H , +. Abbreviations: Ac-Phe-tlRNA, N-acetylphenylalanyl-tRNA; Ac-Phe-puromycin, N-acetylphenylalanylpuromycin.
Biochim. Biophys. Acta, 247 (1971) 304-309
RIBOSOMAL PEPTIDYL TRANSFER REACTION
305
It has been shown that the reaction between Ac-Phe-tRNA and puromycin occurs readily on salt-washed 7o-S ribosomes in the presence of poly(U), Mg2+, and monovalent cation such as K + or NH4+ (refs. 5, 6). Using this system, we have previously found that ribosomes from the mutants of E. coli having an altered specific 5o-S ribosomal protein component (5o-8), have lower peptidyl transferase activity than those from parent QI3 cells at ordinary salt concentration 7. These ribosomes "less active" in peptidyl transferase activity have been shown to be convertible to the "active" state by increasing the environmental K + (or NH4+ ) concentration. To obtain further insight into the activity of ribosomes in the peptidyl transfer reaction, we describe here the Ieaction of puromycin with Ac-Phe-tRNA on ribosomes reconstituted from 3o-S and 5o-S ribosomal subunits. The results suggest that the function of 5o-S ribosomal subunits is affected by the concentration of N H , + more specifically than by that of Mg2+, and indicate that the chemical structure of the 50-8 ribosomal protein component is closely correlated with ribosomal peptidyl transferase activity.
MATERIALS AND METHODS
Ribosomes were prepared according to the method of NIRENBERG8 from E. coli QI3 and E. coli QE2oI (erythromycin-resistant mutant derived from QI3 as
described previouslyg). Ribosomal subunits were prepared by sucrose gradient centrifugation after dissociation by the addition of phosphate 1°, and were dialyzed against o.oi M Tris buffer (pH 7.4) containing o.oi M magnesium acetate. Ac-[a4C]Phe-tRNA was prepared by the method of LAPIDOT et al. 11. Puromycin • 2HCI was purchased from the Nutritional Biochemical Corp. and poly(U) was obtained from C. F. Boehringer and Sohn GmbH. [14C]Phenylalanine (specific radioactivity 455/~C//~mole) was obtained from Schwarz BioResealch Inc. The assay for the formation of Ac-E14C]Phe-puromycin was a modification of the method described by LEDER AND BURSZTYN12 for the assay of N-formylmethionylpuromycin. The reaction conditions are specified in the legends to the tables and figure. After incubation the reaction was stopped by the addition of 0.25 ml of 0. 4 IV[ sodium acetate (pH 5.5), then extracted with 1.5 ml of ethyl acetate with shaking. After centrifugation at 2000 rev./min for IO min, I ml of the ethyl acetate layer was transferred to a scintillation vial containing IO ml of BRAY'Sls solution and radioactivity was counted in a Beckman liquid scintillation counter.
RESULTS AND DISCUSSION
Although it was reported by MONRO1 that the reaction of puromycin with Nblocked aminoacyloligonucleotide fragment from tRNA takes place on 5o-S ribosomal subunits alone in the presence of alcohol, it is evident from Table I, that both ribosomal subunits are required for the formation of Ac-Phe-puromycin in this system. Table I also shows that, as expected from previous work employing 70-S ribosomes 14, Ac-Phe-puromycin was formed at o ° when the preincubation of ribosomal subunits was carried out in the presence of sufficient Mg 2+ and NH4+. Biochim. Biophys. Acta, 247 (1971) 3o4-3o9
306 TABLE
H. TERAOKA, K. TANAKA I
Ac-Phe-PUROMYClN
FORMATION DEPENDING
UPON BOTH RIBOSOMAL SUBUNITS
Expt. I: A m i x t u r e (Ioo #1) contained 4 °/2g of poly(U), 0.72 Ase 0 nm unit of Ac-[I*C~Phe-tRNA (12 ooo c o u n t s / m i n ) and indicated a m o u n t s of 3o-S s u b u n i t s in a s t a n d a r d buffer (5 ° mM T r i s HC1, p H 7.8, 16 mM m a g n e s i u m acetate, 0.2 M NH4C1 ). A suspension of 5o-S s u b u n i t s (ioo/~1) contained indicated a m o u n t s of 5o-S s u b u n i t s in the s t a n d a r d buffer. The m i x t u r e containing 3o-S s u b u n i t s and the suspension of 5o-S s u b u n i t s were p r e i n c u b a t e d at 37" for io nlin, cooled in ice for 5 rain, t h e n combined t o g e t h e r at o °. The p u r o m y c i n reaction (225/,1) was s t a r t e d b y the addition of o . I / * m o l e p u r o m y c i n (dissolved in the s t a n d a r d buffer) to the combined mixture, a n d the reaction t u b e was incubated at o ° for io min. The a s s a y for A c - P h e - p u r o m y c i n formation was carried o u t as described u n d e r MATERIALS AND M E T H O D S , Expt. 2: E x p e r i m e n t a l conditions were the same as described in E x p t . I, except t h a t after preincubation, the m i x t u r e containing 3o-S s u b u n i t s a n d the suspension of 5o-S were combined together w i t h o u t cooling in ice, kept at 37 ° for 3 rain, t h e n cooled to o °.
Expt.
3o-S subunits (A2~0 nm units)
5o-S subunits (A,6o nmUnits)
Ac-Phe-puromycin /ormed (counts/rain)
I
o 0.64 0.32 0.64
1.28 o 0.64 1.28
26 45 585 116o
2
0.64
1.28
1264
It is of interest to note that almost the same level of Ac-Phe-puromycin formation was observed whether the combination of 5o-S and 3o-S subunits of ribosomes was carried out at o ° or 37 ° (Table I, Expts. I and 2). This fact may indicate that association of the two ribosomal subunits takes place even at o °. To obtain further information on the effect of different preincubation conditions on the ability of each subunit to participate in Ac-Phe-puromycin formation, reactions were carried out in which the composition of the reaction mixture and the reaction temperature (o°) were kept constant, any differences in the amounts of Ac-Phe-puromycin formed thus being mainly due to differences in the preincubation conditions. TABLE II ~EEBCT OF TEMPERATURE
IN THE PREINCUBATION
STEP
E x p e r i m e n t a l conditions were the same as described in Table I, E x p t . I, except t h a t the m i x t u r e containing 3o-S s u b u n i t s and t h e suspension of 5o-S s u b u n i t s contained 0.66 A,~ 0 nm unit of 3o-S s u b u n i t s and I.O6 A ~60 nm units of 5o-S subunits, respectively, p r e i n c u b a t e d at the indicated temperatures,
Preineubation temperature Mixture containing 3o-S subunits
Suspension o[ 5o-S subunits
Ac-Phe-puromycin [ormed (counts~rain)
37 ° 37 ° o°
37 ° °Q 37 °
908 47 399
0°
0°
18
As shown in Table I1, preincubation of 5o-S subunits must be carried out at a suitable temperature (37 °) for the subsequent puromycin reaction. When the preBiochim. Biophys. Aeta, 247 (1971) 3o4-3o9
307
RIBOSOMAL PEPTIDYL TRANSFER REACTION
incubation was carried out at low temperature (o°), 5o-S subunits showed no significant activity even if other conditions, such as environmental ionic conditions, were sufficient. When a mixture containing 3o-S subunits, poly(U) and Ac-Phe-tRNA was preincubated at o °, however, significant but not maximum synthesis of AcPhe-puromycin was observed. This fact suggests that the 3o-S subunit preparation used in this study still retains the ability to bind Ac-Phe-tRNA at o °. As already reported 5,6,15, the Ac-Phe-puromycin forming activity of 7o-S ribosomes in the presence of poly(U) is strongly dependent upon the concentration of Mg ~+ and monovalent cations such as K + or NH4+, maximum activity being obtained with lO-2O mlV[ Mg 2. and o.16-o.2 M NH4+. When 5o-S subunits were preincubated in the medium depleted of K + or NH4+, no detectable formation of Ac-Phepuromycin was observed at o ° even in the presence of sufficient NH4+ in the reaction mixture (Table III). Considered together with the results listed in Table II, the pep-
TABLE
Ill
E F F E C T OF N H 4 +
CONCENTRATION
IN T H E P E E I N C U B A T I O N
MEDIUM
P r e i n c u b a t i o n conditions were the same as described in Table I, E x p t . I, except t h a t the m i x t u r e containing 3o-S s u b u n i t s and the suspension of 5o-S s u b u n i t s contained 0.53 A,e 0 nm u n i t of 3o-S s u b u n i t s and 1.o2 A2e0 nm units of 5o-S subunits, respectively, p r e i n c u b a t e d at 37 ° in a m e d i u m containing 16 mM Mg 2+ and indicated concentrations of NH~ +. Prior to the p u r o m y c i n reaction, the ionic concentration of each combined m i x t u r e was adjusted to the same value (o.2 M NH4+, 16 mM Mg2+), t h e n o . I / * m o l e p u r o m y c i n (dissolved in the s t a n d a r d buffer) was added to m a k e a final v o l u m e of 225/21. O t h e r conditions were the same as described in Table I, E x p t . I.
NHa + concentration (M) I n the mixture containing 3o-S subunits
I n the suspension o / 5 o - S subunits
0.2 0.2 o o
0.2 o 0.2 o
A c-Phe-puromycin /ormed (counts~rain)
778 26 75 ° 17
tidyl transferase function of 5o-S ribosomal subunits appears to be activated during the preincubation step in the presence of sufficient NH4 +, the activation rate being largely dependent upon the preincubation temperature. These results are consistent with the suggestion of MISKIN et al. 3,4 that the fragment reaction catalyzed b y ribosomes is largely dependent upon the conformation of ribosomes, which is reversible depending on the K + (or NH4+ ) concentration. The preincubation of 5o-S ribosomal subunits at very low Mg2+ concentration caused no detectable decrease in Ac-Phepuromycin formation (Table IV). This observation strongly supports our previous finding that the affinity of ribosomes (5o-S subunits) for erythromycin is dependent on the environmental concentration of K + or NH4 + but not on that of Mg2+ (see refs. 16, I7) , this being in marked contrast to the cation dependency of preincubation mixtures containing 3o-S subunits. These facts suggest that the function of 5o-S ribosomal subunits in the process of peptide bond formation in ribosomes is, in contrast to that of 3o-S subunits, affected b y the concentration of monovalent cations such as K + or NH~ + more specifically than it is by the concentration of Mg2+. Biochim. Biophys. Acta, 247 (1971) 3o4-3o9
308
H. TERAOKA, K. TANAKA
TABLE IV E F F E C T OF ~V[g2+ CONCENTRATION IN THE PRI~INCUBATION MEDIUM
P r e i n c u b a t i o n conditions were the same as described in Table I, E x p t . i, except t h a t the m i x t u r e containing 3o-S s u b u n i t s and the suspension of 5o-S s u b u n i t s contained o.66 A~60 nm unit of 3o-S and 1.o6 A,e 0 nm units of 5o-S subunits, respectively, p r e i n c u b a t e d at 37 ° in a m e d i u m containing o.2 1V[ NH4+ and indicated concentrations of Mg 2+. O t h e r conditions were the same as described in Table I I I .
Mg 2+ concentration (raM) In the mixture containing 3o-S subunits
In the suspension o/5o-S subunits
16.o 16.o 2.0 2.0
16.o 2.5 16.o 2.5
A c- Phe-puromycin [orrned (counts/min)
846 895 285 254
We have previously shown that the ribosomes from the erythromycin resistant mutants of E. coli, having an altered specific 5o-S ribosomal protein component (5o-8), have distinctly lowered activity in Ac-Phe-puromycin formation, although their ability to bind Ae-Phe-tRNA is more or less unaltered ~. The Ac-Phe-puromycin forming activity of these ribosomes increased very sharply with increase in environmental K + or NH4 + concentration, and reached the same maximum level as that observed for parent QI3 ribosomes at sufficient concentration of these monovalent cations. From these facts, we suggested that a structural change in a protein component influences the conformation of 5o-S ribosomal subunit and induces consequent alteration of the peptidyl transferase activity. To obtain further insight into this, hybrid 7o-S ribosomes were obtained from 3o-S and 5o-S ribosomal subunits prepared from QI3 and its mutant (QE2oI), and the ability of such hybrid ribosomes to function in Ac-Phe-puromycin formation was then tested. As shown by the data in Fig. I, hybrid ribosomes containing QE2oI 5o-S subunits have much lower activity than hybrid ribosomes containing QI 3 5o-S subunits. E_ 1~ 100C
E
~
c_. 5oc
....
./. KCI (raM)
NHaC[ (mM)
Fig.
I. A c - P h e - p u r o m y c i n forming activity of h y b r i d ribosomes obtained from ribosomal subu n i t s of Q I 3 and its m u t a n t . The reaction m i x t u r e contained the following c o m p o n e n t s in a final v o l u m e of 125 #1; 5 ° mlV~ Tris-tiC1 (pH 7.8), 16 mM m a g n e s i u m acetate, o.42 A260 nm unit of Ac-E14C]Phe-ttZNA (93oo counts/rain), 4 ° / z g of poly(U), 0. 4 A2~0 nm unit of 3o-S subunits, o.8 M260 nm u n i t of 5o-S subunits, o. 4 mM puromycin, and a specified concentration of K + or NHa+. I n c u b a t i o n was carried o u t at 37 ° for io min. The assay for A c - P h e - p u r o m y c i n f o r m a t i o n was carried o u t as described u n d e r MATERIALS AND METHODS. O - - O , Q E 2 o I 3o-S s u b u n i t s and Q I 3 5o-S subunits. O - - - O , QI3 3o-S s u b u n i t s and Q E 2 o I 5o-S subunits.
Biochim. Biophys. Acta, 247 (1971) 304-309
309
RIBOSOMAL PEPTIDYL TRANSFER REACTION
R e c e n t l y i t w a s f o u n d t h a t t h e c h a r a c t e r of t h e a l t e r e d 50-8 p r o t e i n c o m p o n e n t is a l w a y s c o - t r a n s d u c e d w i t h t h e e r y t h r o m y c i n r e s i s t a n c e f r o m d o n o r t o recip i e n t b a c t e r i a b y P l k c p h a g e TM. I t w a s f u r t h e r s h o w n t h a t a l t e r a t i o n of 50-8 p r o t e i n c o m p o n e n t i n r i b o s o m e s f r o m t h e s e t r a n s d u c t a n t s is a l w a y s a c c o m p a n i e d b y r e d u c e d r i b o s o m a l p e p t i d y l t r a n s f e r a s e a c t i v i t y TM. T o g e t h e r w i t h t h e r e s u l t s i n Fig. I, t h e s e r e s u l t s g i v e s t r o n g e v i d e n c e t h a t t h e c h e m i c a l s t r u c t u r e of t h e 50-8 r i b o s o m a l p r o t e i n c o m p o p e n t is c l o s e l y c o r r e l a t e d w i t h t h e p e p t i d e s y n t h e s i z i n g a b i l i t y of r i b o s o m e s .
ACKNOWLEDGMENT W e t h a n k Mr. M. T a m a k i f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e .
t~EFERENCES i 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 I8 19
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Biochim. Biophys. Acta, 247 CI97I) 304-309