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Interaction of aminoacyl-tRNA synthetases with ribosomes and ribosomal subunits
175
Biochimica et Biophysica Acta, 4 2 5 ( 1 9 7 6 ) 1 7 5 - - 1 8 4 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 98519
INTERACTION OF AMINOACYL-tRNA SYNTHETASES WITH RIBOSOMES AND RIBOSOMAL SUBUNITS
HERMANN GRAF *
National Institutes of Health, National Heart and Lung Institute, Bethesda, Md. (U.S.A.) (Received A u g u s t 2 7 t h , ( 1 9 7 5 )
Summary Salt-washed ribosomes from rabbit reticulocytes stimulate seven partially purified aminoacyl-tRNA synthetases up to threefold: arginyl-, alanyl-, isoleucyl-, lysyl-, methionyl-, phenylalanyl- and valyl-tRNA synthetase. Both isolated subunits but not total ribosomal RNA show the stimulation. The increase in synthetase activity is also found in a highly purified Escherichia coli system employing homogenous (E. coli)phenyl-alanyl-tRNA synthetase and (E. coli)t R N A P h e. The biological significance of the stimulation is discussed.
Introduction
Amino acid activation and peptide bond formation in animal cells have previously been considered to take place at separate locations inside the cell, i.e. cytoplasm and rough endoplasmic reticulum. Recent observations suggest that aminoacyl-tRNA synthetases existing in complex form are associated with ribosomes and tRNA, or even that all components of the protein-synthesizing machinery are assembled into a large complex structure [1--6]. The finding that ribosomes contain synthetase activity, which had been considered merely as a contamination, is now discussed as representing the remainder o f a labile, intracellular structure partially broken-down by the isolation procedure [1,7]. The work presented here is an attempt to demonstrate that there is also a functional interaction between ribosomes and aminoacyl-tRNA synthetases in rabbit reticulocytes and E. coli. Since it is difficult to prove whether association of components o f the cell structure found after disrupture of the cell is
* Present address: Institut ffir Physiologische Chemie, Physikalische B i o c h e m i e und Zellbiologie der Universit~/t M£inchen, D 8000 Mllnchen 2, Goethestrasse 33, G.F.R.
176 not an artifact of the isolation procedure, the functional approach reported here provides an independent way of looking into the relation between the different components. Part of this work has appeared in abstract form [8]. Materials and Methods (1) Radioactive amino acids were purchased from Amersham Searle, [ a 2p]. p y r o p h o s p h a t e from New England Nuclear. Rabbit liver t R N A was obtained from General Biochemicals, Chagrin Falls, Ohio, and purified t R N A eh e from E. coli from Miles Laboratories Inc. The t R N A accepted 957o phenylalanine and 5% leucine. DEAE-, CM- and P-cellulose were purchased from Whatman and pretreated as described by the manufacturer, Sepharose 4B and Sephadex G-200 were from Pharmacia.
(2) Enzymes Mammalian phenylalanyl-tRNA synthetase was isolated from the crude preparation o f initiation factor M~ obtained from the 0.5 M KC1 wash of rabbit reticulocyte ribosomes by fractionation on DEAE-cellulose [9]. 10 ml of the 35--60% a m m o n i u m sulfate fractionated IF-M ~ (approx. 40 mg/ml) was loaded on to a Sephadex G-200 column (2.5 X 90 cm) equilibrated with 20 mM Tris/HC1 p H 7.5, 1 mM dithiothreitol, 0.1 mM EDTA and 100 mM KC1. The synthetase was eluted with the same buffer at approximately 15 ml/h and 5-ml fractions were collected. 20-pl aliquots were assayed for synthetase activity; the peak of activity eluted shortly after the void volume, clearly separated from initiation factor M~. Active fractions were pooled, concentrated by ultrafiltration {UM-10 membrane of Amicon Corp.) and stored in 100-pl aliquots in liquid nitrogen. Methionyl-tRNA synthetase {rabbit r e t i c u l o c y t e ) w a s obtained from the supernatant fraction prepared as described by Crystal et al. [ 1 0 ] , 840 ml of supernatant were made 20 mM in Tris/HCl pH 7.5 and fractionated by adding 147 g neutralised a m m o n i u m sulfate to yield 30% saturation. After stirring for 1 h the mixture was centrifuged {20 min, 15 000 X g), the pellet discarded and the supernatant further precipitated by adding 136 g ammonium sulfate to obtain 557o saturation. After stirring for 1 h the mixture was centrifuged as above and the pellet was dissolved in a minimum of buffer A (20 mM Tris/HC1 pH 7.5, 1 mM dithiothreitol, 20 mM KC1, 0.1 mM EDTA and 107o glycerol). This fractionation resulted in an approximately sevenfold increase of specific activity with a yield of 50%. Methionyl-tRNA synthetase was further purified by applying 5 ml of the 30--557o fraction (90 mg protein/ml) to a Sepharose 4B column (2.6 X 30 cm) equilibrated with buffer A. The column was eluted with the same buffer at 15 ml/h and 3-ml fractions were collected. 3-~1 aliquots were assayed for methionyl-tRNA synthetase activity, active fractions pooled and concentrated by ultrafiltration (PM-30 membrane, Amicon Corp.). The concentrated preparation was stored in 100-~1 aliquots in liquid nitrogen. The methionyl-tRNA synthetase activity eluted at about two void volumes well separated from the bulk of hemoglobin. This procedure resulted in a five-fold increase of specific activity with approximately 357o yield. (spec. act. 1.64 units/pg, 1 unit = 1 pmol t R N A acylated per min). Attempts to purify further methionyl-tRNA synthetase on
177 DEAE-ceUulose, DEAE-Sephadex, P-cellulose, CM-cellulose and hydroxyapatite resulted in a considerable loss of enzyme activity. The source of alanyl-, arginyl-, lysyl-, isoleucyl- and valyl-synthetase was a crude preparation obtained from rabbit reticulocyte supernatant by treatment with DEAE-cellulose and kindly donated by Dr. D. Picciano. This preparation was shown to contain all the t w e n t y aminoacyl-tRNA synthetases and proved highly active in in vitro hemoglobin synthesis. The highly purified phenylalanyl-tRNA syr~thetase from E. coli was a generous gift of Dr. A. B6ck, Regensburg, Germany. On sodium dodecyl sulfate-gel electrophoresis this preparation showed one band accounting for more than 95% of the protein applied. Ribosomes and ribosomal subunits Crude ribosomes from rabbit reticulocyte lysate were obtained as described [10]. The pellet of crude ribosomes was suspended by gentle rubbing with a glass rod in buffered sucrose (0.25 M sucrose, 10 mM Tris/HC1 pH 7.5, 1 mM dithiothreitol, 3 mM MgC12, 0.1 mM EDTA) and diluted to a concentration of 100--200 A260 nm units/ml. Salt washing of the ribosomes was achieved by adding solid KC1 to a final concentration of 0.5 M. After stirring for 30 min the suspension was centrifuged in a Ti 65 R o t o r (Beckman) for 2 h at 65 000 rev./min in a Spinco ultracentrifuge. In some experiments a sucrose cushion (5 ml 1 M sucrose plus the above ions per centrifuge tube) was used. The ribosomal pellet was suspended as described above and 100-pl aliquots stored in liquid nitrogen. High salt ribosomal subunits 7-ml washed ribosomes (2100-A260 nm units) were diluted to 20 ml with 20 mM Tris/HC1 pH 7.5, 1 mM dithiothreitol, 3 mM MgC12 0.5 M KC1 and incubated for 6 min at 37°C. 10 ml of the above buffer was added and the solution was layered on a 15--40% convex exponential sucrose gradient in the above buffer containing 0.3 M KC1 in a Ti 14 zonal rotor (Beckman). An overlay of 70 ml was added and the gradient centrifuged for 3.5 h at 45 000 rev./min. The gradient was unloaded by pumping 40% sucrose from the edge at 10 ml/min and ~ m l fractions were collected. The absorption at 260 nm was monitored in a flow-through p h o t o m e t e r (Isco). Fractions containing the 40-S and 60-S subunits and the 80-S monosomes were pooled and concentrated by centrifugation for 14 h in a Ti 60 rotor (Beckman) at 60 000 rev./min. The pellets were suspended in buffered sucrose containing 50 mM KC1 and stored in 50-#1 aliquots in liquid nitrogen. The recovery was 54 A260 nm units of 40-S subunits, 112 A260 nm units of 60-S subunits and 605 A260 nm units of monosomes. "Run o f f " ribosomal subunits L y s a t e ribosomes were prepared as in Ref. 10 except that the lysing solution contained 3 mM MgC12. The pellet was suspended in buffered sucrose containing 50 mM KCI. 1000 A260 nm units were incubated in a 40 ml reaction mixture containing 3.6 ml of rabbit reticulocyte supernatant for 40 rain at 37°C. The concentration of salts, ATP, GTP, phosphoenolpyruvate, pyruvate
178 kinase, 20 non-radioactive amino acids and rabbit reticulocyte tRNA was as in the endogenous mRNA-dependent globin-synthesizing system [10]. 5 ml of 4 M KC1 was added to yield a final concentration of 0.5 M and the subunits separated on a sucrose gradient in a zonal rotor as described above. No extra washing step with high salt was included in the procedure. The yield was 88 As 60 am units of 40-S subunits and 325 A260 nm units of 60-S subunits. Only a small shoulder in the As 60 nm tracing in the region of monosomes could be seen. The purity o f the 60-S subunits was checked by gel electrophoresis of the extracted RNA. 350 pl of 60-S subunits were thrice extracted with 1 ml watersaturated phenol containing 1% sodium dodecyl sulfate. The aqueous extract was precipitated with two vol. of 95% ethylalcohol at --20°C for 15 h. The precipitate was collected by centrifugation and dissolved in water. 2 pl (0.5 A260 nm units) of this solution were analysed on a 2.2% polyacrylamide gel run at 5 m A / t u b e for 75 min. Scanning of the gel at 260 nm revealed a major peak of 28 S RNA and some small peaks of lower molecular weight. The gel was calibrated by running RNA standards in parallel and comparing the distances of migration on the scan. Contamination of the 28 S RNA from the 60-S subunits in the region of 18 S was less than 5% and a peak of 5 S RNA could be clearly identified. The subunits showed activity in the poly(U) assay comparable to ribosomes prepared by the standard m e t h o d [10]. Total activity with both subunits was more than t w e n t y f o l d greater than the level obtained with the 60 S subunit alone. Rabbit reticulocyte tRNA was isolated from the supernatant fraction as described [ 11].
Assays Aminoacyl-tRNA synthetase activity was measured in 50-pl reaction mixtures containing 100 mM Tris/HC1 pH 7.0, 1 mM dithiothreitol, 2 mM ATP, 40 pM 14C-labelled L-amino acids (spec. act. 50--100 Ci/Mol) and the Mg 2÷ concentration optimal for the specific enzyme. These optima were determined for the enzymes from rabbit reticulocytes: alanyl-tRNA synthetase 7 mM; arginyl-, 15 mM; isoleucyl-, 6 mM; lysyl-, 7 mM; methionyl-, 5 mM and valyl-tRNA synthetase 10 mM. Phenylalanyl-tRNA synthetase from E. coli was measured at 10 mM MgC12. t R N A was present in saturating amounts, 1--2 A260 nm units per reaction mixture of rabbit reticulocyte or rabbit liver tRNA, 0.09 A260 am units per reaction mixture of purified (E. coli)tRNA Phe. The standard reaction was run for 10 min at 37°C and stopped by adding 3 ml cold 10% trichloracetic acid. After 10 min at 0°C the precipitate was collected on a Mfllipore filter, washed three times with 3 ml cold 5% trichloroacetic acid, dried and counted in a TriCarb (Packard) liquid scintillation counter (counting efficiency 80%). No correction for background radioactivity was made except when indicated in the figure legends. The activity of ribosomes and ribosomal subunits in protein synthesis was measured in a poly(U)-dependent system using [ 1 , C ] p h e n y l a l a n y l . t R N A as substrate as described [10]. Incubation was for 2 rain at 37°C in the presence of 4 mM Mg 2÷ and saturating amounts of initiation and elongation factors.
179 Results
Crude ribosomes obtained by centrifugation of a lysate from rabbit reticulocytes at 100 000 × g contain a considerable amount of aminoacyl-tRNA synthetases. After washing of a suspension of these ribosomes with 0.5 M KC1 and repelleting, most of the synthetase activity is removed from the ribosomes and recovered in the supernatant. Such salt~washed ribosomes were added to a standard aminoacylation reaction mixture containing saturatin~ amounts of crude t R N A and partially purified methionyl-tRNA synthetase, from rabbit reticulocytes, [ ' 4 C ] methionine, ATP and salts as described in Materials and Methods. The results obtained are shown in Fig. 1: the a m o u n t of radioactive material precipitable with cold trichloroacetic acid increased proportional with the amount of ribosomes added until a level approximately two times that with t R N A synthetase alone was reached. Ribosomes alone accounted for less than 10% of total incorporated [ ' 4 C] methionine. Similar results were obtained with partially purified valyl-tRNA synthetase from rabbit reticulocytes and [ ~4 C] valine (not shown). The increase in precipitable counts in this system reaches a
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Fig. 1. A c t i o n of washed rabbit retieuloeyte and E. c o l i ribosomes on the homologous a m i n o a c y l - t R N A synthetase. The conditions o f the reaction and the assay procedure were as described in materials and methods. I n the retieuloeyte system 1.4 units o f partially purified m e t ~ o n y i - t R N A synthetase per reaction mtxtuze was used; in the E. coli system 1,1 units highly purified p h e n y i a l a n y l - t R N A synthetase was employed per reaction x~XtULre. A f t e r t h e standard i n c u b a t i o n at 37°C, 0.5 A 2 6 0 r m t units o f rabbit llver t R N A was added t o the latter system before precipitation w i t h tdehloraeetie acid. I n b o t h systems one set o f reaction ntixtuzes contained ldbosomes alone Ln the amaounts indicated (rabbit r e t i c u l o c y t e (e); E. c o l i (o)), another set contained Hbosomes plus enzyme (rabbit retienioeyte (a); E. coli (D)). F ~ . 2. Mg 2+ dependence o f L - v a l y i - t R N A synthetase in the presence and absence o f ribosomes. Assay c o n d i t i o n s w e r e as d e s c r i b e d i n m a t e r i a l and m e t h o d s . T h e r e a c t i o n m i x t t t r e s c o n t a i n e d 2 u n i t s e n z y m e (a), 1.3 A 2 6 0 n m u n i t s salt w a s h e d r i b o s o m e s (e) o r i d e n t i c a l a m o u n t s of r i b o s o m e s p l u s e n z y m e (m). The Mg 2+ c o n c e n t r a t i o n was as i n d i c a t e d . A set of r e a c t i o n s m i x t u r e s c o n t a i n i n g a n i d e n t i c a l a m o u n t o f r i b o s o m e s p l u s e n z y m e w a s i n c u b a t e d for 1 0 rain at 9 0 ° C a f t e r t h e a d d i t i o n o f t r i c h l o r o a c e t i c acid ( T C A ) (o). E n z y m e , r i b o s o m e s a n d t R N A w e r e derived f r o m r a b b i t r e t i c u l o e y t e s .
180 level three times higher than that of the reaction with synthetase alone. Adding albumin to a standard reaction mixture did not affect the reaction (not shown). The increase of trichloroacetic acid preCipitable radioactive material by adding ribosomes was found over a wide range of Mg 2÷ (Fig. 2) or spermine concentration (not shown). This indicates that ribosomes did not cause stimulation by shifting the actual Mg2÷ or spermine concentration to the o p t i m u m by either introducing these ions into the reaction which had been previously bound to them, or reducing the concentration in the reaction mixture by binding Mg 2÷ or spermine. When the reaction mixture containing enzyme plus ribosomes was treated with 10% trichloroacetic acid at 90°C for 10 min after the standard incubation at 37°C trichloroacetic acid-precipitable radioactivity is reduced to background level (Fig. 2), t h a t means no [~ 4C]valine had been incorporated into protein. To prove further the absence of protein synthesis, an analysis of the product of the reaction with [ ~4 C] methionine was performed. Two reaction mixtures, ten times the standard assay volume, one containing enzyme alone and the other enzyme plus ribosomes were incubated for 15 min at 37°C and precipitated with cold 10% trichloroacetic acid. The precipitate was washed with 5-ml portions of 10% trichloroacetic acid until less than 0.2% o f the total precipitable counts were found in the wash solution. The precipitate was then dissolved in 0.5 M ammonia, fl-mercaptoethanol added to a final concentration of 140 mM, and hydrolysed for 1 h at 37°C. The solution was neutralised with HC1, clarified by centrifugation, and the hydrolysate subjected to high-voltage paper electrophoresis as described [12]. In both samples 85--90% of the applied radioactivity ran with a methionine marker. Only background activity was found at the locations of markers of methionyl-valine, methionyl-methionine, methionyl-valyl-histidine and methionyl-valyl-leucine. 10--15% o f the radioactivity applied ran as an unidentified spot in the opposite direction, whereas almost no radioactivity remained at the origin. A time curve of the synthetase reaction in the presence of ribosomes is given in Fig. 3. Three reaction mixtures ten times the standard assay (0.5 ml) conraining either ribosomes or enzyme alone or ribosomes plus enzyme were incubated at 37 °C. Aliquots were removed at the times indicated and treated as in the standard assay. The curve obtained with ribosomes plus enzyme had an approximately three times greater slope than the curve with enzyme alone, demonstrating a higher velocity of the stimulated reaction. The washed ribosomes were functionally intact as shown in Table I. They were active in poly{U)-dependent synthesis of poly{Phe) at low Mg2÷ concentration in the presence of saturating amounts of initiation and elongation factors. Table I also shows the effect of sequential washing of the ribosomes upon their activity in protein synthesis and their capacity to stimulate the aminoacylation reaction. With the first washing the bulk of synthetase activity was removed and the remaining activity gradually decreased to less than 1% of the initial by further washing and centrifugation through a sucrose cushion. On the other hand, activity in protein synthesis and capacity to stimulate aminoacylation were only reduced to about 50%. With the unwashed ribosomes no stimulation can be observed because of the large background of contaminating synthetase activity. Stimulation of the aminoacyl-tRNA synthetase reaction by
181
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Fig. 3. T i m e d e p e n d e n c e o f t h e a m i n o a c y l a t i o n r e a c t i o n . E x p e r i m e n t a l details ~re g i v e n in t h e t e x t . R e a c t i o n m i x t u r e s c o n t a i n e d 1.3 A 2 6 0 n m u n i t s o f r i b o s o m e s o n l y (o), 2 u n i t s v a l y l - t R N A s y n t h e t a s e o n l y (4), or t h e s a m e a m o u n t o f r i b o s o m e s plus e n z y m e as in t h e o t h e r t w o r e a c t i o n s Ca). Fig. 4. T h e e f f e c t o f r i b o s o m a l s u b u n i t s f r o m r a b b i t r e t i c u l o c y t e s o n v a l y l - t R N A s y n t h e t a s e . R e a c t i o n c o n d i t i o n s w e r e as d e s c r i b e d in m a t e r i a l s a n d m e t h o d s . O n e set o f r e a c t i o n c o n t a i n e d 40-S s u b u n i t s (A) o r 60-S s u b u n i t s a l o n e ( e ) in t h e a m o u n t s i n d i c a t e d , o n e set c o n t a i n e d 1 u n i t p a r t i a l l y p u r i f i e d v a l y l - t R N A s y n t h e t a s e f r o m r a b b i t r e t i c u l o c y t e s plus t h e s u b u n i t s , 1 A 2 6 0 n m u n i t o f 40-S s u b u n i t s w a s a s s u m e d t o b e 58 p m o l , 1 A 2 6 0 n m u n i t o f 60-S s u b u n i t s t o b e 23 p m o l .
TABLE I A C T I V I T Y O F R I B O S O M E S IN S Y N T H E S I S O F V A L Y L - t R N A FORMATION OF POLYPHENYLALANINE
A N D IN P O L Y ( U ) - D E P E N D E N T
R i b o s o m e s w e r e w a s h e d b y stirring in 0 . 5 M K C I f o r 3 0 r a i n o r f o r 15 h as d e s c r i b e d in Materials a n d M e t h o d s . T h e y w e r e p e l l e t e d e i t h e r d i r e c t l y or t h r o u g h a 1 M s u c r o s e c u s h i o n . A c y l a t l o n a n d p o l y ( P h e ) s y n t h e s i s w a s m e a s u r e d as d e s c r i b e d in Materials a n d M e t h o d s . R e a c t i o n m i x t u r e s f o r a c y l a t i o n c o n t a i n e d e i t h e r 1.3 A 2 6 0 n m u n i t s of r i b o s o m e s (first c o l u m n ) o r 3.3 u n i t s v a l y l - t R N A s y n t h e t a s e a l o n e ( s e c o n d c o l u m n ) o r t h e s a m e a m o u n t o f b o t h ( c o l u m n d e s i g n a t e d " r i b o s o m e s a n d e n z y m e " ) . T h e v a l u e s for p o l y ( P h e ) f o r m a t i o n w e r e t a k e n f r o m t h e l i n e a r p a r t o f a c u r v e o b t a i n e d b y i n c r e a s i n g t h e a m o u n t of r i b o s o m e s p r e s e n t in t h e p r o t e i n s y n t h e s i s r e a c t i o n m i x t u r e . Ribosomes
Unwashed 30-min wash (sucrose cushion) 2 X 30-rain wash 2 × 15-h w a s h 3 X 30-min wash
Acylation (pmol [14C]valine b o u n d to t R N A / m i n )
Poly(U)~lependent formation of poly(Phe) (pmol poly(Phe) synthesized/mln A 260nm unit of ribosomes)
Ribosomes
Enzyme
Ribosomes + enzyme
29.1
--
--
5.3
2.5 2.5 2.5 2.5
23.9 14.7 16.6 12.5
3.8 3.8
0.63 0.40 0.40 0.23
5.5
182 washed ribosomes occurs not only with methionyl- and valyl-tRNA synthetases but with all the synthetases assayed as shown in Table II. No a t t e m p t was undertaken to investigate all t w e n t y synthetases. Stimulation still occurs when rabbit reticulocyte tRNA is replaced by rabbit liver tRNA. The same holds true when rabbit reticulocyte phenylalanyl-tRNA synthetase and tRNA are replaced by the corresponding E. coli components (heterologous system, data not shown). The stimulation of aminoacylation can also be demonstrated in a highly purified E. coli system. Fig. 1 gives the results obtained with a more than 95% pure preparation of phenylalanyl-tRNA synthetase from E. coli and pure (E. c o l i ) t R N A Phe. Again with increasing amounts of washed ribosomes added to the reaction mixture, a proportional increase in synthetase activity is observed. When ribosomal RNA isolated from either 80-S monosomes from rabbit reticulocytes or from 60-S ribosomal subunits is added to the aminoacylation reaction in amounts corresponding to those of ribosomes used in the earlier experiments no stimulation of activity is found (data not shown). Ribosomal subunits isolated either by "run o f f " or treatment with 0.5 M KC1 (high salt subunits) both stimulated the incorporation of [14C]valine (Fig. 4 ) a n d [14C]methio_ nine in trichloracetic acid-precipitable material. Endogenous synthetase activity of the isolated subunits was less than 10% o f the enzyme activity used in the standard reaction. Similar results were obtained with isolated monosomes. Whether there is a difference in the a m o u n t of stimulation between 40-S and 60-S subunits and 80-S monosomes cannot be determined from the present data. Synthetase activity can also be measured by determining the a m o u n t of 32p exchanged from pyrophosphate into ATP. This reaction reflects the reversible formation of an aminoacyl-adenylate which is considered an intermediate in the aminoacylation reaction. With most synthetases this reaction is independent of the homologous tRNA. The effect of synthetase-free ribosomes on the ATP-PPi exchange reaction was measured in a system employing partially purified valyl-tRNA synthetase and salt-washed ribosomes both from rabbit reticulocytes. The reaction was performed as described [13], and the optimal salt
T A B L E II S T I M U L A T I O N O F D I F F E R E N T S Y N T H E T A S E S BY R A B B I T R E T I C U L O C Y T E R I B O S O M E S T h e a s s a y c o n d i t i o n s w e r e as d e s c r i b e d in Material a n d M e t h o d s . T h e r e a c t i o n m i x t u r e s c o n t a i n e d 0 . 1 5 - - 0 . 5 ~tl p a r t i a l l y p u r i f i e d s y n t h e t a s e f r o m r a b b i t r e t i c u l o c y t e s a n d a n a m o u n t o f w a s h e d r i b o s o m e s o p t i m a l for s t i m u l a t i o n ( 1 . 0 - - 1 . 5 A 2 6 0 n m u n i t s ) . N u m b e r s r e p r e s e n t p m o l o f 14C.iabclled a m i n o a c i d s b o u n d t o t R N A p e r 1 0 rain. Amino acid
Enzyme
Ribosomes
Ribosomes + enzyme
Valine Lysine Arginine Phenylalanine Methionine Alanine Isoleucine
24.9 15.1 16.0 10.3 17.2 15.3 15,1
3.4 8.7 6.8 4.1 2.4 0.7 4.8
77.7 51.7 44.6 42.5 36.3 22.0 28.5
183 concentrations determined for the reticulocyte enzymes. When increasing amounts of ribosomes were added under these conditions to a constant level of valyl-tRNA synthetase an increase in exchange activity occurred, whereas ribosomes alone showed a low level of activity (data not shown). Similar results were obtained, when L-phenylalanine-dependent pyrophosphate exchange was measured using partially purified phenylalanyl-tRNA synthetase from reticulocytes. Discussion
Activation of amino acids by aminoacyl-tRNA synthetases in the cytoplasm and peptide synthesis on the ribosomes are consecutive steps in protein biosynthesis. It is advantageous for the cell when the components necessary for performing consecutive steps of a biochemical pathway are physically assembled in one compartment. There is evidence for binding of synthetases to ribosomes and ribosomal subunits (refs. 1--6 and Graf, H., unpublished) at least in Eucaryotes. The data presented here suggest in addition a functional interaction. When washed ribosomes with low endogenous synthetase activity are added to various aminoacyl-tRNA synthetases in homologous or heterologous systems, an increase in trichloracetic acid-precipitable counts occurs. A trivial explanation for this increase, namely a change in the actual Mg2÷ or spermine concentration resulting from the addition of the ribosomes is ruled out by demonstrating the stimulation over a wide range of concentration of these ions. Three lines of evidence suggest that the stimulation does not result from protein synthesis. (1) Treatment of the reaction mixture with hot trichloroacetic acid reduces precipitable radioactivity to background level. (2) Analysis of the product obtained with methionyl-tRNA synthetase yields only methionine. No radioactivity in the di- and tripeptides which would be synthesized when rabbit globin message is used as a template is found. (3) The ATP-PPi exchange catalysed by valyl- and phenylalanyl-tRNA synthetase is also stimulated by ribosomes. Both of the isolated ribosomal subunits from rabbit reticulocytes as well as 80-S monosomes produce the stimulation, whereas isolated ribosomal RNA has no influence upon the synthesis of valyl-tRNA. The investigation of the effect of isolated total ribosomal proteins on the synthetase reaction has been complicated by solubility problems. Preliminary results suggest that they are not stimulatory. The results obtained in the heterologous system show that the small amount of reticulocyte synthetase remaining on the washed ribosomes is not necessary for stimulation: Gilbert and Anderson [11] have shown that rabbit reticulocyte phenylalanyl-tRNA synthetase does not acylate (E. coli)tRNA but it is shown here that stimulation of the E. coli enzyme by reticulocyte ribosomes does occur. It is unlikely that stimulation is caused by damaged ribosomes because they are active in poly(U)-coded poly(Phe) synthesis. However, the possibility cannot be ruled out that there is a mixed population of ribosomes, part of which is active in protein synthesis whilst the other part is stimulatory. The results obtained in the highly purified E. coli system demonstrate that a
184 contaminating factor is not causing the stimulation. An inhibitor o f synthetase activity present in the partially purified enzyme preparations, for example, could be inactivated by the ribosomes. This would also result in an increase of enzyme activity. Repeated washing with high salt neither destroys the capacity to stimulate acylation nor the activity of in vitro protein synthesis of the ribosomes. Thus the ribosomal or subunit structure itself, or a factor tightly b o u n d to it is responsible for the stimulation. Since the seven aminoacyl-tRNA synthetases tested can be stimulated by ribosomes it seems reasonable to assume that also the other enzymes react in the same way. If ribosomes would protect acylated t R N A against deacylation an increase in aminoacylation would also be found. But this explanation for the stimulation of synthetase activity by ribosomes is rendered unlikely by the finding that ATP-PPi exchange is stimulated too. Therefore it is suggested that the ribosomes or subunits affect the synthetase molecule itself. The binding of synthetases to ribosomes and the stimulation of synthetase activity by ribosomes are both believed to be a means of increasing the efficiency of the protein synthesizing machinery inside the cell. Loftfield [14] calculated from data obtained in vitro with E. coli lysyl-tRNA synthetase and the activity necessary for protein synthesis in rapidly growing cells, that the in vitro activity is five times lower than that required in vivo. The observed stimulation of synthetases in the presence of ribosomes could well account for this differeric e.
Acknowledgements This work was supported by a fellowship from the Deutsche Forschungsgemeinschaft. I wish to thank Dr. W.F. Anderson in whose laboratory most of this work was done for hospitality and encouragement. Drs. J. R o b e r t s o n and R. Streeck contributed suggestions during the preparation of the manuscript. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
B a n d y o p a d h y a y , A . K . a n d D e u t s c h e r , M.P. ( 1 9 7 1 ) J. Mol. Biol. 6 0 , 1 1 3 - - 1 2 2 V e n n e g o o r , C. a n d B l o e m e n d a l , H. ( 1 9 7 2 ) EVLr. J . B i o c h e m . 2 6 , 4 6 2 - - 4 7 3 H i l d e r m a n , R . H . a n d D e u t s c h e r , M.P. ( 1 9 7 4 ) J . Biol. C h e m . 2 4 9 , 5 3 4 6 - - 5 3 4 8 Irvin, J . D . a n d H a r d e s t y , B. ( 1 9 7 2 ) B i o c h e m i s t r y 1 1 , 1 9 1 5 - - 1 9 2 0 S o m , K . a n d H a r d e s t y , B. ( 1 9 7 5 ) A r c h . B i o c h e m . B i o p h y s . 1 6 6 , 5 0 7 - - 5 1 7 R o b e r t s , W . K . a n d C o l e m a n , W . H . ( 1 9 7 2 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 4 6 , 2 0 6 - - 2 1 4 Kisselev, L . L . a n d F a v o r o v a , O . O . ( 1 9 7 4 ) in A d v a n c e s in E n z y m o l o g y ( M e i s t e r , A., e d . ) , Vol. 4 0 , p p . 141--238, John Wiley and Sons, New York G r a f , H. ( 1 9 7 3 ) F e d . P r o c . 3 2 , A b s t r . 4 5 9 M e r r i c k , W., G r a f , H. and A n d e r s o n , W . F . ( 1 9 7 4 ) i n M e t h o d s in E n z y m o l o g y ( M o l d a v e , K . and G r o s s m a n , L., eds.), Vol. 3 0 , p p . 1 2 8 - - 1 3 6 , A c a d e m i c Press, N e w Y o r k C r y s t a l , R . G . , E l s o n , N . A . a n d A n d e r s o n , W . F . ( 1 9 7 4 ) in M e t h o d s in E n z y m o l o g y ( M o l d a v e , K . a n d G r o s s m a n , L., eds.), V o L 3 0 , p p . 1 0 1 - - 1 2 7 , A c a d e m i c Press, N e w Y o r k G i l b e r t , J . M . and A n d e r s o n . W . F . ( 1 9 7 0 ) J. Biol. C h e m . 2 4 5 , 2 3 4 2 - - 2 3 4 9 E l s o n , N . A . , A d a m s , S . L . , M e r r i c k , W.C., S a f e r , B. and A n d e r s o n , W . F . ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 3074--3079 R o t h , J . R . a n d A m e s , B.N. ( 1 9 6 6 ) J. Mol. Biol. 2 2 , 3 2 5 - - 3 3 4 L o f t f i e l d , R . B . ( 1 9 7 2 ) in Progress in N u c l e i c A c i d R e s e a r c h and M o l e c u l a r B i o l o g y ( D a v i d s o n , J . N . and C o h n , W.E.° eds.), Vol. 1 2 , p p . 8 7 - - 1 2 8 , A c a d e m i c Press, N e w Y o r k
Biochimica et Biophysica Acta, 4 2 5 ( 1 9 7 6 ) 1 7 5 - - 1 8 4 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 98519
INTERACTION OF AMINOACYL-tRNA SYNTHETASES WITH RIBOSOMES AND RIBOSOMAL SUBUNITS
HERMANN GRAF *
National Institutes of Health, National Heart and Lung Institute, Bethesda, Md. (U.S.A.) (Received A u g u s t 2 7 t h , ( 1 9 7 5 )
Summary Salt-washed ribosomes from rabbit reticulocytes stimulate seven partially purified aminoacyl-tRNA synthetases up to threefold: arginyl-, alanyl-, isoleucyl-, lysyl-, methionyl-, phenylalanyl- and valyl-tRNA synthetase. Both isolated subunits but not total ribosomal RNA show the stimulation. The increase in synthetase activity is also found in a highly purified Escherichia coli system employing homogenous (E. coli)phenyl-alanyl-tRNA synthetase and (E. coli)t R N A P h e. The biological significance of the stimulation is discussed.
Introduction
Amino acid activation and peptide bond formation in animal cells have previously been considered to take place at separate locations inside the cell, i.e. cytoplasm and rough endoplasmic reticulum. Recent observations suggest that aminoacyl-tRNA synthetases existing in complex form are associated with ribosomes and tRNA, or even that all components of the protein-synthesizing machinery are assembled into a large complex structure [1--6]. The finding that ribosomes contain synthetase activity, which had been considered merely as a contamination, is now discussed as representing the remainder o f a labile, intracellular structure partially broken-down by the isolation procedure [1,7]. The work presented here is an attempt to demonstrate that there is also a functional interaction between ribosomes and aminoacyl-tRNA synthetases in rabbit reticulocytes and E. coli. Since it is difficult to prove whether association of components o f the cell structure found after disrupture of the cell is
* Present address: Institut ffir Physiologische Chemie, Physikalische B i o c h e m i e und Zellbiologie der Universit~/t M£inchen, D 8000 Mllnchen 2, Goethestrasse 33, G.F.R.
176 not an artifact of the isolation procedure, the functional approach reported here provides an independent way of looking into the relation between the different components. Part of this work has appeared in abstract form [8]. Materials and Methods (1) Radioactive amino acids were purchased from Amersham Searle, [ a 2p]. p y r o p h o s p h a t e from New England Nuclear. Rabbit liver t R N A was obtained from General Biochemicals, Chagrin Falls, Ohio, and purified t R N A eh e from E. coli from Miles Laboratories Inc. The t R N A accepted 957o phenylalanine and 5% leucine. DEAE-, CM- and P-cellulose were purchased from Whatman and pretreated as described by the manufacturer, Sepharose 4B and Sephadex G-200 were from Pharmacia.
(2) Enzymes Mammalian phenylalanyl-tRNA synthetase was isolated from the crude preparation o f initiation factor M~ obtained from the 0.5 M KC1 wash of rabbit reticulocyte ribosomes by fractionation on DEAE-cellulose [9]. 10 ml of the 35--60% a m m o n i u m sulfate fractionated IF-M ~ (approx. 40 mg/ml) was loaded on to a Sephadex G-200 column (2.5 X 90 cm) equilibrated with 20 mM Tris/HC1 p H 7.5, 1 mM dithiothreitol, 0.1 mM EDTA and 100 mM KC1. The synthetase was eluted with the same buffer at approximately 15 ml/h and 5-ml fractions were collected. 20-pl aliquots were assayed for synthetase activity; the peak of activity eluted shortly after the void volume, clearly separated from initiation factor M~. Active fractions were pooled, concentrated by ultrafiltration {UM-10 membrane of Amicon Corp.) and stored in 100-pl aliquots in liquid nitrogen. Methionyl-tRNA synthetase {rabbit r e t i c u l o c y t e ) w a s obtained from the supernatant fraction prepared as described by Crystal et al. [ 1 0 ] , 840 ml of supernatant were made 20 mM in Tris/HCl pH 7.5 and fractionated by adding 147 g neutralised a m m o n i u m sulfate to yield 30% saturation. After stirring for 1 h the mixture was centrifuged {20 min, 15 000 X g), the pellet discarded and the supernatant further precipitated by adding 136 g ammonium sulfate to obtain 557o saturation. After stirring for 1 h the mixture was centrifuged as above and the pellet was dissolved in a minimum of buffer A (20 mM Tris/HC1 pH 7.5, 1 mM dithiothreitol, 20 mM KC1, 0.1 mM EDTA and 107o glycerol). This fractionation resulted in an approximately sevenfold increase of specific activity with a yield of 50%. Methionyl-tRNA synthetase was further purified by applying 5 ml of the 30--557o fraction (90 mg protein/ml) to a Sepharose 4B column (2.6 X 30 cm) equilibrated with buffer A. The column was eluted with the same buffer at 15 ml/h and 3-ml fractions were collected. 3-~1 aliquots were assayed for methionyl-tRNA synthetase activity, active fractions pooled and concentrated by ultrafiltration (PM-30 membrane, Amicon Corp.). The concentrated preparation was stored in 100-~1 aliquots in liquid nitrogen. The methionyl-tRNA synthetase activity eluted at about two void volumes well separated from the bulk of hemoglobin. This procedure resulted in a five-fold increase of specific activity with approximately 357o yield. (spec. act. 1.64 units/pg, 1 unit = 1 pmol t R N A acylated per min). Attempts to purify further methionyl-tRNA synthetase on
177 DEAE-ceUulose, DEAE-Sephadex, P-cellulose, CM-cellulose and hydroxyapatite resulted in a considerable loss of enzyme activity. The source of alanyl-, arginyl-, lysyl-, isoleucyl- and valyl-synthetase was a crude preparation obtained from rabbit reticulocyte supernatant by treatment with DEAE-cellulose and kindly donated by Dr. D. Picciano. This preparation was shown to contain all the t w e n t y aminoacyl-tRNA synthetases and proved highly active in in vitro hemoglobin synthesis. The highly purified phenylalanyl-tRNA syr~thetase from E. coli was a generous gift of Dr. A. B6ck, Regensburg, Germany. On sodium dodecyl sulfate-gel electrophoresis this preparation showed one band accounting for more than 95% of the protein applied. Ribosomes and ribosomal subunits Crude ribosomes from rabbit reticulocyte lysate were obtained as described [10]. The pellet of crude ribosomes was suspended by gentle rubbing with a glass rod in buffered sucrose (0.25 M sucrose, 10 mM Tris/HC1 pH 7.5, 1 mM dithiothreitol, 3 mM MgC12, 0.1 mM EDTA) and diluted to a concentration of 100--200 A260 nm units/ml. Salt washing of the ribosomes was achieved by adding solid KC1 to a final concentration of 0.5 M. After stirring for 30 min the suspension was centrifuged in a Ti 65 R o t o r (Beckman) for 2 h at 65 000 rev./min in a Spinco ultracentrifuge. In some experiments a sucrose cushion (5 ml 1 M sucrose plus the above ions per centrifuge tube) was used. The ribosomal pellet was suspended as described above and 100-pl aliquots stored in liquid nitrogen. High salt ribosomal subunits 7-ml washed ribosomes (2100-A260 nm units) were diluted to 20 ml with 20 mM Tris/HC1 pH 7.5, 1 mM dithiothreitol, 3 mM MgC12 0.5 M KC1 and incubated for 6 min at 37°C. 10 ml of the above buffer was added and the solution was layered on a 15--40% convex exponential sucrose gradient in the above buffer containing 0.3 M KC1 in a Ti 14 zonal rotor (Beckman). An overlay of 70 ml was added and the gradient centrifuged for 3.5 h at 45 000 rev./min. The gradient was unloaded by pumping 40% sucrose from the edge at 10 ml/min and ~ m l fractions were collected. The absorption at 260 nm was monitored in a flow-through p h o t o m e t e r (Isco). Fractions containing the 40-S and 60-S subunits and the 80-S monosomes were pooled and concentrated by centrifugation for 14 h in a Ti 60 rotor (Beckman) at 60 000 rev./min. The pellets were suspended in buffered sucrose containing 50 mM KC1 and stored in 50-#1 aliquots in liquid nitrogen. The recovery was 54 A260 nm units of 40-S subunits, 112 A260 nm units of 60-S subunits and 605 A260 nm units of monosomes. "Run o f f " ribosomal subunits L y s a t e ribosomes were prepared as in Ref. 10 except that the lysing solution contained 3 mM MgC12. The pellet was suspended in buffered sucrose containing 50 mM KCI. 1000 A260 nm units were incubated in a 40 ml reaction mixture containing 3.6 ml of rabbit reticulocyte supernatant for 40 rain at 37°C. The concentration of salts, ATP, GTP, phosphoenolpyruvate, pyruvate
178 kinase, 20 non-radioactive amino acids and rabbit reticulocyte tRNA was as in the endogenous mRNA-dependent globin-synthesizing system [10]. 5 ml of 4 M KC1 was added to yield a final concentration of 0.5 M and the subunits separated on a sucrose gradient in a zonal rotor as described above. No extra washing step with high salt was included in the procedure. The yield was 88 As 60 am units of 40-S subunits and 325 A260 nm units of 60-S subunits. Only a small shoulder in the As 60 nm tracing in the region of monosomes could be seen. The purity o f the 60-S subunits was checked by gel electrophoresis of the extracted RNA. 350 pl of 60-S subunits were thrice extracted with 1 ml watersaturated phenol containing 1% sodium dodecyl sulfate. The aqueous extract was precipitated with two vol. of 95% ethylalcohol at --20°C for 15 h. The precipitate was collected by centrifugation and dissolved in water. 2 pl (0.5 A260 nm units) of this solution were analysed on a 2.2% polyacrylamide gel run at 5 m A / t u b e for 75 min. Scanning of the gel at 260 nm revealed a major peak of 28 S RNA and some small peaks of lower molecular weight. The gel was calibrated by running RNA standards in parallel and comparing the distances of migration on the scan. Contamination of the 28 S RNA from the 60-S subunits in the region of 18 S was less than 5% and a peak of 5 S RNA could be clearly identified. The subunits showed activity in the poly(U) assay comparable to ribosomes prepared by the standard m e t h o d [10]. Total activity with both subunits was more than t w e n t y f o l d greater than the level obtained with the 60 S subunit alone. Rabbit reticulocyte tRNA was isolated from the supernatant fraction as described [ 11].
Assays Aminoacyl-tRNA synthetase activity was measured in 50-pl reaction mixtures containing 100 mM Tris/HC1 pH 7.0, 1 mM dithiothreitol, 2 mM ATP, 40 pM 14C-labelled L-amino acids (spec. act. 50--100 Ci/Mol) and the Mg 2÷ concentration optimal for the specific enzyme. These optima were determined for the enzymes from rabbit reticulocytes: alanyl-tRNA synthetase 7 mM; arginyl-, 15 mM; isoleucyl-, 6 mM; lysyl-, 7 mM; methionyl-, 5 mM and valyl-tRNA synthetase 10 mM. Phenylalanyl-tRNA synthetase from E. coli was measured at 10 mM MgC12. t R N A was present in saturating amounts, 1--2 A260 nm units per reaction mixture of rabbit reticulocyte or rabbit liver tRNA, 0.09 A260 am units per reaction mixture of purified (E. coli)tRNA Phe. The standard reaction was run for 10 min at 37°C and stopped by adding 3 ml cold 10% trichloracetic acid. After 10 min at 0°C the precipitate was collected on a Mfllipore filter, washed three times with 3 ml cold 5% trichloroacetic acid, dried and counted in a TriCarb (Packard) liquid scintillation counter (counting efficiency 80%). No correction for background radioactivity was made except when indicated in the figure legends. The activity of ribosomes and ribosomal subunits in protein synthesis was measured in a poly(U)-dependent system using [ 1 , C ] p h e n y l a l a n y l . t R N A as substrate as described [10]. Incubation was for 2 rain at 37°C in the presence of 4 mM Mg 2÷ and saturating amounts of initiation and elongation factors.
179 Results
Crude ribosomes obtained by centrifugation of a lysate from rabbit reticulocytes at 100 000 × g contain a considerable amount of aminoacyl-tRNA synthetases. After washing of a suspension of these ribosomes with 0.5 M KC1 and repelleting, most of the synthetase activity is removed from the ribosomes and recovered in the supernatant. Such salt~washed ribosomes were added to a standard aminoacylation reaction mixture containing saturatin~ amounts of crude t R N A and partially purified methionyl-tRNA synthetase, from rabbit reticulocytes, [ ' 4 C ] methionine, ATP and salts as described in Materials and Methods. The results obtained are shown in Fig. 1: the a m o u n t of radioactive material precipitable with cold trichloroacetic acid increased proportional with the amount of ribosomes added until a level approximately two times that with t R N A synthetase alone was reached. Ribosomes alone accounted for less than 10% of total incorporated [ ' 4 C] methionine. Similar results were obtained with partially purified valyl-tRNA synthetase from rabbit reticulocytes and [ ~4 C] valine (not shown). The increase in precipitable counts in this system reaches a
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,
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Fig. 1. A c t i o n of washed rabbit retieuloeyte and E. c o l i ribosomes on the homologous a m i n o a c y l - t R N A synthetase. The conditions o f the reaction and the assay procedure were as described in materials and methods. I n the retieuloeyte system 1.4 units o f partially purified m e t ~ o n y i - t R N A synthetase per reaction mtxtuze was used; in the E. coli system 1,1 units highly purified p h e n y i a l a n y l - t R N A synthetase was employed per reaction x~XtULre. A f t e r t h e standard i n c u b a t i o n at 37°C, 0.5 A 2 6 0 r m t units o f rabbit llver t R N A was added t o the latter system before precipitation w i t h tdehloraeetie acid. I n b o t h systems one set o f reaction ntixtuzes contained ldbosomes alone Ln the amaounts indicated (rabbit r e t i c u l o c y t e (e); E. c o l i (o)), another set contained Hbosomes plus enzyme (rabbit retienioeyte (a); E. coli (D)). F ~ . 2. Mg 2+ dependence o f L - v a l y i - t R N A synthetase in the presence and absence o f ribosomes. Assay c o n d i t i o n s w e r e as d e s c r i b e d i n m a t e r i a l and m e t h o d s . T h e r e a c t i o n m i x t t t r e s c o n t a i n e d 2 u n i t s e n z y m e (a), 1.3 A 2 6 0 n m u n i t s salt w a s h e d r i b o s o m e s (e) o r i d e n t i c a l a m o u n t s of r i b o s o m e s p l u s e n z y m e (m). The Mg 2+ c o n c e n t r a t i o n was as i n d i c a t e d . A set of r e a c t i o n s m i x t u r e s c o n t a i n i n g a n i d e n t i c a l a m o u n t o f r i b o s o m e s p l u s e n z y m e w a s i n c u b a t e d for 1 0 rain at 9 0 ° C a f t e r t h e a d d i t i o n o f t r i c h l o r o a c e t i c acid ( T C A ) (o). E n z y m e , r i b o s o m e s a n d t R N A w e r e derived f r o m r a b b i t r e t i c u l o e y t e s .
180 level three times higher than that of the reaction with synthetase alone. Adding albumin to a standard reaction mixture did not affect the reaction (not shown). The increase of trichloroacetic acid preCipitable radioactive material by adding ribosomes was found over a wide range of Mg 2÷ (Fig. 2) or spermine concentration (not shown). This indicates that ribosomes did not cause stimulation by shifting the actual Mg2÷ or spermine concentration to the o p t i m u m by either introducing these ions into the reaction which had been previously bound to them, or reducing the concentration in the reaction mixture by binding Mg 2÷ or spermine. When the reaction mixture containing enzyme plus ribosomes was treated with 10% trichloroacetic acid at 90°C for 10 min after the standard incubation at 37°C trichloroacetic acid-precipitable radioactivity is reduced to background level (Fig. 2), t h a t means no [~ 4C]valine had been incorporated into protein. To prove further the absence of protein synthesis, an analysis of the product of the reaction with [ ~4 C] methionine was performed. Two reaction mixtures, ten times the standard assay volume, one containing enzyme alone and the other enzyme plus ribosomes were incubated for 15 min at 37°C and precipitated with cold 10% trichloroacetic acid. The precipitate was washed with 5-ml portions of 10% trichloroacetic acid until less than 0.2% o f the total precipitable counts were found in the wash solution. The precipitate was then dissolved in 0.5 M ammonia, fl-mercaptoethanol added to a final concentration of 140 mM, and hydrolysed for 1 h at 37°C. The solution was neutralised with HC1, clarified by centrifugation, and the hydrolysate subjected to high-voltage paper electrophoresis as described [12]. In both samples 85--90% of the applied radioactivity ran with a methionine marker. Only background activity was found at the locations of markers of methionyl-valine, methionyl-methionine, methionyl-valyl-histidine and methionyl-valyl-leucine. 10--15% o f the radioactivity applied ran as an unidentified spot in the opposite direction, whereas almost no radioactivity remained at the origin. A time curve of the synthetase reaction in the presence of ribosomes is given in Fig. 3. Three reaction mixtures ten times the standard assay (0.5 ml) conraining either ribosomes or enzyme alone or ribosomes plus enzyme were incubated at 37 °C. Aliquots were removed at the times indicated and treated as in the standard assay. The curve obtained with ribosomes plus enzyme had an approximately three times greater slope than the curve with enzyme alone, demonstrating a higher velocity of the stimulated reaction. The washed ribosomes were functionally intact as shown in Table I. They were active in poly{U)-dependent synthesis of poly{Phe) at low Mg2÷ concentration in the presence of saturating amounts of initiation and elongation factors. Table I also shows the effect of sequential washing of the ribosomes upon their activity in protein synthesis and their capacity to stimulate the aminoacylation reaction. With the first washing the bulk of synthetase activity was removed and the remaining activity gradually decreased to less than 1% of the initial by further washing and centrifugation through a sucrose cushion. On the other hand, activity in protein synthesis and capacity to stimulate aminoacylation were only reduced to about 50%. With the unwashed ribosomes no stimulation can be observed because of the large background of contaminating synthetase activity. Stimulation of the aminoacyl-tRNA synthetase reaction by
181
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Fig. 3. T i m e d e p e n d e n c e o f t h e a m i n o a c y l a t i o n r e a c t i o n . E x p e r i m e n t a l details ~re g i v e n in t h e t e x t . R e a c t i o n m i x t u r e s c o n t a i n e d 1.3 A 2 6 0 n m u n i t s o f r i b o s o m e s o n l y (o), 2 u n i t s v a l y l - t R N A s y n t h e t a s e o n l y (4), or t h e s a m e a m o u n t o f r i b o s o m e s plus e n z y m e as in t h e o t h e r t w o r e a c t i o n s Ca). Fig. 4. T h e e f f e c t o f r i b o s o m a l s u b u n i t s f r o m r a b b i t r e t i c u l o c y t e s o n v a l y l - t R N A s y n t h e t a s e . R e a c t i o n c o n d i t i o n s w e r e as d e s c r i b e d in m a t e r i a l s a n d m e t h o d s . O n e set o f r e a c t i o n c o n t a i n e d 40-S s u b u n i t s (A) o r 60-S s u b u n i t s a l o n e ( e ) in t h e a m o u n t s i n d i c a t e d , o n e set c o n t a i n e d 1 u n i t p a r t i a l l y p u r i f i e d v a l y l - t R N A s y n t h e t a s e f r o m r a b b i t r e t i c u l o c y t e s plus t h e s u b u n i t s , 1 A 2 6 0 n m u n i t o f 40-S s u b u n i t s w a s a s s u m e d t o b e 58 p m o l , 1 A 2 6 0 n m u n i t o f 60-S s u b u n i t s t o b e 23 p m o l .
TABLE I A C T I V I T Y O F R I B O S O M E S IN S Y N T H E S I S O F V A L Y L - t R N A FORMATION OF POLYPHENYLALANINE
A N D IN P O L Y ( U ) - D E P E N D E N T
R i b o s o m e s w e r e w a s h e d b y stirring in 0 . 5 M K C I f o r 3 0 r a i n o r f o r 15 h as d e s c r i b e d in Materials a n d M e t h o d s . T h e y w e r e p e l l e t e d e i t h e r d i r e c t l y or t h r o u g h a 1 M s u c r o s e c u s h i o n . A c y l a t l o n a n d p o l y ( P h e ) s y n t h e s i s w a s m e a s u r e d as d e s c r i b e d in Materials a n d M e t h o d s . R e a c t i o n m i x t u r e s f o r a c y l a t i o n c o n t a i n e d e i t h e r 1.3 A 2 6 0 n m u n i t s of r i b o s o m e s (first c o l u m n ) o r 3.3 u n i t s v a l y l - t R N A s y n t h e t a s e a l o n e ( s e c o n d c o l u m n ) o r t h e s a m e a m o u n t o f b o t h ( c o l u m n d e s i g n a t e d " r i b o s o m e s a n d e n z y m e " ) . T h e v a l u e s for p o l y ( P h e ) f o r m a t i o n w e r e t a k e n f r o m t h e l i n e a r p a r t o f a c u r v e o b t a i n e d b y i n c r e a s i n g t h e a m o u n t of r i b o s o m e s p r e s e n t in t h e p r o t e i n s y n t h e s i s r e a c t i o n m i x t u r e . Ribosomes
Unwashed 30-min wash (sucrose cushion) 2 X 30-rain wash 2 × 15-h w a s h 3 X 30-min wash
Acylation (pmol [14C]valine b o u n d to t R N A / m i n )
Poly(U)~lependent formation of poly(Phe) (pmol poly(Phe) synthesized/mln A 260nm unit of ribosomes)
Ribosomes
Enzyme
Ribosomes + enzyme
29.1
--
--
5.3
2.5 2.5 2.5 2.5
23.9 14.7 16.6 12.5
3.8 3.8
0.63 0.40 0.40 0.23
5.5
182 washed ribosomes occurs not only with methionyl- and valyl-tRNA synthetases but with all the synthetases assayed as shown in Table II. No a t t e m p t was undertaken to investigate all t w e n t y synthetases. Stimulation still occurs when rabbit reticulocyte tRNA is replaced by rabbit liver tRNA. The same holds true when rabbit reticulocyte phenylalanyl-tRNA synthetase and tRNA are replaced by the corresponding E. coli components (heterologous system, data not shown). The stimulation of aminoacylation can also be demonstrated in a highly purified E. coli system. Fig. 1 gives the results obtained with a more than 95% pure preparation of phenylalanyl-tRNA synthetase from E. coli and pure (E. c o l i ) t R N A Phe. Again with increasing amounts of washed ribosomes added to the reaction mixture, a proportional increase in synthetase activity is observed. When ribosomal RNA isolated from either 80-S monosomes from rabbit reticulocytes or from 60-S ribosomal subunits is added to the aminoacylation reaction in amounts corresponding to those of ribosomes used in the earlier experiments no stimulation of activity is found (data not shown). Ribosomal subunits isolated either by "run o f f " or treatment with 0.5 M KC1 (high salt subunits) both stimulated the incorporation of [14C]valine (Fig. 4 ) a n d [14C]methio_ nine in trichloracetic acid-precipitable material. Endogenous synthetase activity of the isolated subunits was less than 10% o f the enzyme activity used in the standard reaction. Similar results were obtained with isolated monosomes. Whether there is a difference in the a m o u n t of stimulation between 40-S and 60-S subunits and 80-S monosomes cannot be determined from the present data. Synthetase activity can also be measured by determining the a m o u n t of 32p exchanged from pyrophosphate into ATP. This reaction reflects the reversible formation of an aminoacyl-adenylate which is considered an intermediate in the aminoacylation reaction. With most synthetases this reaction is independent of the homologous tRNA. The effect of synthetase-free ribosomes on the ATP-PPi exchange reaction was measured in a system employing partially purified valyl-tRNA synthetase and salt-washed ribosomes both from rabbit reticulocytes. The reaction was performed as described [13], and the optimal salt
T A B L E II S T I M U L A T I O N O F D I F F E R E N T S Y N T H E T A S E S BY R A B B I T R E T I C U L O C Y T E R I B O S O M E S T h e a s s a y c o n d i t i o n s w e r e as d e s c r i b e d in Material a n d M e t h o d s . T h e r e a c t i o n m i x t u r e s c o n t a i n e d 0 . 1 5 - - 0 . 5 ~tl p a r t i a l l y p u r i f i e d s y n t h e t a s e f r o m r a b b i t r e t i c u l o c y t e s a n d a n a m o u n t o f w a s h e d r i b o s o m e s o p t i m a l for s t i m u l a t i o n ( 1 . 0 - - 1 . 5 A 2 6 0 n m u n i t s ) . N u m b e r s r e p r e s e n t p m o l o f 14C.iabclled a m i n o a c i d s b o u n d t o t R N A p e r 1 0 rain. Amino acid
Enzyme
Ribosomes
Ribosomes + enzyme
Valine Lysine Arginine Phenylalanine Methionine Alanine Isoleucine
24.9 15.1 16.0 10.3 17.2 15.3 15,1
3.4 8.7 6.8 4.1 2.4 0.7 4.8
77.7 51.7 44.6 42.5 36.3 22.0 28.5
183 concentrations determined for the reticulocyte enzymes. When increasing amounts of ribosomes were added under these conditions to a constant level of valyl-tRNA synthetase an increase in exchange activity occurred, whereas ribosomes alone showed a low level of activity (data not shown). Similar results were obtained, when L-phenylalanine-dependent pyrophosphate exchange was measured using partially purified phenylalanyl-tRNA synthetase from reticulocytes. Discussion
Activation of amino acids by aminoacyl-tRNA synthetases in the cytoplasm and peptide synthesis on the ribosomes are consecutive steps in protein biosynthesis. It is advantageous for the cell when the components necessary for performing consecutive steps of a biochemical pathway are physically assembled in one compartment. There is evidence for binding of synthetases to ribosomes and ribosomal subunits (refs. 1--6 and Graf, H., unpublished) at least in Eucaryotes. The data presented here suggest in addition a functional interaction. When washed ribosomes with low endogenous synthetase activity are added to various aminoacyl-tRNA synthetases in homologous or heterologous systems, an increase in trichloracetic acid-precipitable counts occurs. A trivial explanation for this increase, namely a change in the actual Mg2÷ or spermine concentration resulting from the addition of the ribosomes is ruled out by demonstrating the stimulation over a wide range of concentration of these ions. Three lines of evidence suggest that the stimulation does not result from protein synthesis. (1) Treatment of the reaction mixture with hot trichloroacetic acid reduces precipitable radioactivity to background level. (2) Analysis of the product obtained with methionyl-tRNA synthetase yields only methionine. No radioactivity in the di- and tripeptides which would be synthesized when rabbit globin message is used as a template is found. (3) The ATP-PPi exchange catalysed by valyl- and phenylalanyl-tRNA synthetase is also stimulated by ribosomes. Both of the isolated ribosomal subunits from rabbit reticulocytes as well as 80-S monosomes produce the stimulation, whereas isolated ribosomal RNA has no influence upon the synthesis of valyl-tRNA. The investigation of the effect of isolated total ribosomal proteins on the synthetase reaction has been complicated by solubility problems. Preliminary results suggest that they are not stimulatory. The results obtained in the heterologous system show that the small amount of reticulocyte synthetase remaining on the washed ribosomes is not necessary for stimulation: Gilbert and Anderson [11] have shown that rabbit reticulocyte phenylalanyl-tRNA synthetase does not acylate (E. coli)tRNA but it is shown here that stimulation of the E. coli enzyme by reticulocyte ribosomes does occur. It is unlikely that stimulation is caused by damaged ribosomes because they are active in poly(U)-coded poly(Phe) synthesis. However, the possibility cannot be ruled out that there is a mixed population of ribosomes, part of which is active in protein synthesis whilst the other part is stimulatory. The results obtained in the highly purified E. coli system demonstrate that a
184 contaminating factor is not causing the stimulation. An inhibitor o f synthetase activity present in the partially purified enzyme preparations, for example, could be inactivated by the ribosomes. This would also result in an increase of enzyme activity. Repeated washing with high salt neither destroys the capacity to stimulate acylation nor the activity of in vitro protein synthesis of the ribosomes. Thus the ribosomal or subunit structure itself, or a factor tightly b o u n d to it is responsible for the stimulation. Since the seven aminoacyl-tRNA synthetases tested can be stimulated by ribosomes it seems reasonable to assume that also the other enzymes react in the same way. If ribosomes would protect acylated t R N A against deacylation an increase in aminoacylation would also be found. But this explanation for the stimulation of synthetase activity by ribosomes is rendered unlikely by the finding that ATP-PPi exchange is stimulated too. Therefore it is suggested that the ribosomes or subunits affect the synthetase molecule itself. The binding of synthetases to ribosomes and the stimulation of synthetase activity by ribosomes are both believed to be a means of increasing the efficiency of the protein synthesizing machinery inside the cell. Loftfield [14] calculated from data obtained in vitro with E. coli lysyl-tRNA synthetase and the activity necessary for protein synthesis in rapidly growing cells, that the in vitro activity is five times lower than that required in vivo. The observed stimulation of synthetases in the presence of ribosomes could well account for this differeric e.
Acknowledgements This work was supported by a fellowship from the Deutsche Forschungsgemeinschaft. I wish to thank Dr. W.F. Anderson in whose laboratory most of this work was done for hospitality and encouragement. Drs. J. R o b e r t s o n and R. Streeck contributed suggestions during the preparation of the manuscript. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
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