Ribosome-catalysed peptidyl transfer: the polyphenylalanine system

J. Mol. Biol. (1968) 35, 333-345

RJbosome-catalysed Peptidyl Transfer: the Polyphenylalanine System B. E. 1~. 1VrADW.Nt,1~. 1~. TRAUT$ AND I~. E. ]~/[Ol~O§

Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, England (Received 20 December 1967) The reaction of puromycin with polyphenylala~ino-charged Esvherichia coli ribosomes has boon characterizod. Release of polyphonylalanino from transfer RNA was estimated by a new, rapid method based on differontial solubilities of substrate and product in m-cresol. Occurrence of a reaction in the absence of supornatant and GTP was demonstrated with (a) polyphonylalaalino-chargod 70 s ribosomes which had boon washed with salt solution under conditions similar to those used for removing the supomatant factors of protein synthesis, and (b) purified, polyphenylala~ine-chargod 50s subunits. Only monovalont and divalent cations and buffer are required for this "supornatant-indopendont" reaction, and under suitablo conditions 70 to 80% of the polyphonylala~ine is released from transfer RNA. Evidence from these and other results indicates that the puromycin reaction takes place by the same mechanism as peptide bond formation in protein synthesis, and that peptide bond formation is catalysed by the 50 s ribosomal subunit and does not directly involve supernatant factors or GTP. The reaction has characteristics typical of enzyme reactions, and the catalytic agent has been named "peptidyl transferase". Kinetics of the supernatant-independent reaction are heterogeneous. A fraction of the charged ribosomes is highly reactive and may represent a preformed enzyme-substrate complex. Effects of puromycin concentration and various other factors on the kinetics are described. The reaction is inhibited by addition of 4 E-urea or 0.5 % sodium dodecyl sulphate, by prior heating of charged ribosomes to 70°C, and by pro-treatment with 0.5 to 2% formaldehyde. The principle findings with 70s ribosomes were confirmed with isolated, polyphenylalanlne-charged 50 s subunits. 1. I n t r o d u c t i o n The value of the polyphenylalanlne system for studies on the mechanism of peptide bond formation became apparent when Gilbert (1963) showed t h a t (a) polyphenylalanine is covalently bound to t R N A H (both during and after synthesis), (b) at low magnesium ion concentrations the polyphenylalanyl-tRNA remains associated with the 50 s subunit, and (c) incubation of polyphenylalanine-charged ribosomes with puromycin results in release of polyphenylalanlne from t R N A . t Reprint requests should be sent to Dr B. E. H. Maden, whose present address is: Department of Biochemistry, Albert Einstein College of Medicine, Eastchester Road and Morris Park Avenue, Bronx, N.Y. 10461, U.S.A. $ Present address: Institut de Biologic Mol6eulaire, Universit6 de Gen6ve, 24 Quai de l'Eeole de Medeeine, Geneva, Switzerland. § Present address: C.S.I.C. Institute de Biologia Celular, Madrid 6, Spain. [I Abbreviations used: tRNA, transfer RNA; SDS, sodium dodecyl sulphate.

383

334

B. E. H. M A D E N , R. R. T R A U T A N D R. E. M O N R O

Following this work, T r a u t & Monte (1964) used the puromycin reaction as a model system for studying peptide bond formation. T h e y observed t h a t the reaction of puromycin with washed, polyphenylalanlne-charged ribosomes is p a r t l y independent of added supernatant factors and GTP, and t h a t the reaction can take place on isolated charged 50 s subunits. I t was concluded t h a t either "(1) a supernatant factnr and G T P are required [for peptide bond formation] b u t an intermediate is formed in which t h e y are tightly and specifically bound to the ribosomes; or (2) supernatant factor and G T P act in a prior step to form a reactive polyphenylalanyl-tRNA-50 s ribosome complex, and the reaction of this intermediate-to form a peptide bond is catalysed b y the ribosome itself". We have subsequently characterized in more detail the reaction which occurs in the absence of added supernatant factors and GTP. We describe here a rapid assay and some general properties of the reaction. Our present findings together with other observations indicate t h a t peptide bond formation is catalysed b y the 50 s ribosome itself. Effects of cations and p H on the reaction are reported in another p a p e r (iYladen & Mouro, manuscript in preparation). P a r t of this work has been briefly reported elsewhere (]¢Ionro, Maden & Traut, 1967). 2. M a t e r i a l s a n d M e t h o d s (a) Ge~l e~ract~

Cell extracts ($30), ribosomes and supernatant ($100) were prepared from frozen ~scher/ch/a co//strain M.R.E. 600 (Cammack & Wade, 1965) b y a method slmilar to that of Matthaci & Nirenberg (1961).

(b) Polyphenylalanine.charged riboaome~ Charged ribosomes were prepared from ribosomes and S100, or from $30, in a system essentially as described by Traut & Monte (1964), but with 0.01 M-magnesium acetate and with 0.1 M-17H4C1 in place of KC1. [14C]Phenylalanine (Amersham)was 0"05rn~ and 10mc/m-mole. Incubation was for 20rnln at 3O°C (longer incubations resulted in appreciable hydrolysis of polyphenylala~Sne from tRNA). The ribosomes were isolated b y centrlfugation, washed 3 times by repeated re-suspension and centrifugation in 0-01 MTris buffer (pH 7.4), 0.01 M-magnesium acetate and 0-5 M-NH4C1, and finally resuspended in 0.01 M-Tris buffer (pH 7.4) and 0.01 M-magnesium acetate. After centrifugation for 10 min at 15,000 g to remove aggregated material, the ribosome solution was stored unfrozen at 0°C until used. Typical incubation resulted in incorporation of 10 to 20 phenylaJA.nlne residues per 70 s ribosome. This value probably under estimates the average chain length since it is likely that not all the ribosomes become charged (Gilbert, 1963; Maden, 1966). I n three typical preparations, the average loss of polyphenylalanlne from tRNA during the washing procedure was of the order of 10% (Maden, 1966). Preparations stored for more than a week as described showed little or no hydrolysis of polyphenylalanyl-tRNA or loss of reactivity towards puromycin. (c) Pobyphenylalanin~.charged 50 S ~bosome~ Charged 70 s ribosomes (unwashed) wore dialysed for 12 hr against 0.01 M-Tris buffer (pH 7.4) and 10 -4 M-magnesium acotato. 1-ml. samples containing 5 mg ribosomos were layered on 20 to 5% sucrose gradients (30 mL) containing the same buffer and centrifuged for 10 hr at 24,000 rov.]mln in the Bockman SW25.1 rotor at 4°C. Fractions wore collected and o.D. (260 m#) and radioactivity moasurod with suitable portions. I n general, 90% of the radioactive material originally prosont on 70 s ribosomes was rocovored with the 50 s subunlts. Peak fractions wore pooled, adjusted to 0.01 M-magnesium acetate and the charged 50 s ribosomes polloted by oontrifugation for 4 hr at 50,000 g, resuspondod in the

THE POLYPHENYLALANINE

SYSTEM

335

b o t t o m 2 ml. o f supernatan% (which contained further unpelleted 50 s subunlts) a n d stored a t 0°C. The carry-over of sucrose into assay m i ~ u r e s was shown n o t to interfere w i t h t h e p u r o m y c i n reaction. Re-centrifugation in sucrose gradients confirmed t h a t t h e charged 50 s subunits were free from 30 s subunits a n d from 70 s ribosomes. (d) Assay o/ ~romycin re~tion (i) Pri~ip~ o$ assay [z4C]Polyphenylalanine-charged ribosomes were incubated w i t h puromyein. The e x t e n t o f reaction was estimated b y the cresol assay, in which excess m-cresol is a d d e d a n d t h e samples are filtered, d r i e d a n d assayed for r a d i o a c t i v i t y . P o l y p h e n y l a l a n y l - t R N A is insoluble a n d is r e t a i n e d b y t h e filters. F r e e polyphenylalanine or polyphenylalanylp u r o m y c i n dissolve a n d pass through. The percentage of polyphenylalanine released from t R N A is therefore given b y t h e difference in r a d i o a c t i v i t y between p u r o m y c i n - t r e a t e d a n d u n t r e a t e d izero time) samples. The percentage release value is n o t necessarily proportional to t h e percentage of polyp h e n y l a n y l - t R N A complexes reacting, since it is possible t h a t polyphenylalanlne chains are of heterogeneous lengths, a n d t h a t different chain lengths react a t different rates. This criticism also applies to t h e SDS m e t h o d (Traut & Monro, 1964) and, in general, to methods studying release of nascent protein. However, "percentage release" provides a measure of relative extents of reaction, a n d is a d e q u a t e for m a n y purposes. (fi) I ~ u b a t ~ c o n d i t / o ~ The s t a n d a r d incubation miYture contained 0.2 m g [140]polyphenylalanine.charged ribosomes (70 s or 50 s)/ml., 0-05 ~-Tris acetate ( p H 7.2 a n d 7.8, respectively, a t 30°C a n d 0°C), 0.01 ~-magnesium acetate a n d 0.1 ~-arnmonium acetate (the same ionic composition as used for polyphenylalanine synthesis). 0" 1-ml. portions were brought to the desired temperature. The reaction was initiated b y addition of puromyein a n d t e r m i n a t e d b y a d d i t i o n (with vigorous V o r t e x mi~ing) of 3 ml. m-cresol. Cooled cresol was used for t~inating 0°C incubations. Incubations of 30 sec or less were stopped within 2 see b y r a p i d freezing, effected b y plunging tubes into an acetone-solid CO~ mixture. The frozen pellets were dispersed in cresol with vigorous mixing.

(iii) Cresol assay procedurs After addition of cresol, tubes were left a t room t e m p e r a t u r e for 15 to 90 min. (Samples m i x e d w i t h cresol can also be stored a t --20°C pending filtration.) I m m e d i a t e l y prior t o filtration, samples were again subjected to Vortex mixing to t a k e u p small deposits of precipitate on the walls of t h e tubes. Samples were filtered t h r o u g h cresol-soaked glass filters ( W h a t m a n GL/C, 2 cm) a t a r a t e o f a b o u t 3 ml./20 see. Tubes were rinsed twice w i t h cresol a n d t h e filters washed once more w i t h cresol a n d twice w i t h 95~o ethanol. F i l t e r s were glued to planchets, dried a t 120°C for a t least 20 rain, a n d counted in a n end-window gas-flow counter a t an efficiency of a b o u t 25 %. Filters with 3H precipitates can be immersed in teluene-based scintillation fluid for scintillation counting. )

(iv) Preca/ut/ons M-Cresol (liquid or vapour) is poisonous. To mi~imi~.e contact, cresol was delivered by suitable dispensers a n d filtration was carried o u t in a fume cupboard, using t h i c k rubbez gloves. Tubes from cresol assays were rinsed w i t h ethanol before washing in t h e n o r m a l manner. Thorough drying of filters is essential, since cresol v a p o u r causes r u p t u r e of end windows in gas-flow counters. iv) Factors affecting cresol assay Polyphenylalanine is insoluble in m-cresol containing more t h a n 10% iv/v) water. A 30-fold excess o f cresol is optimal. Recovery o f r a d i o a c t i v i t y on parallel cresol a n d h o t trichloroacefi¢ acid assays of freshly charged ribosomes (at 1 m g riboecmes/ml.) agree within 10%. This shows t h a t recovery o f p o l y p h e n y l a l a n y l - t R N A is satisfactory a n d t h a t t h e polyphenylalanine is n e a r l y all a t t a c h e d to t R N A . A t low ribosome concentration, where precipitates are v e r y small, recovery o f

336

B.E.H.

M A D E N , R . R. T R A U T A N D R. E . M O N R 0

radioactive m a t e r i a l is slightly decreased. Taking 1 m g ribosomes/ml, as giving 100% recovery, 0.2 rng/ml, gives a b o u t 85% a n d 0.I mg/ml, gives 70% recovery. 0.2 m g of ribosomes/ml, was used in most experiments, since t h e p u r o m y c i n reaction was somewhat less r a p i d a t higher ribosome concentrations. (This effect was n o t investigated in detail.) The 15% loss of recovery should n o t affect t h e calculation o f percentage reaction, since t h e ribosome concentration was constant t h r o u g h o u t a n y given experiment. Controls showed t h a t recovery of p o l y p h e n y l a l a n y l - t R N A was unaffected under all conditions employed here a n d b y Maden & Monro (manuscript in preparation), except t h a t a t high Mg 2+ (1 M) or Be n+ (0.01 M) concentrations recovery was a p p a r e n t l y greater. (Therefore under these conditions separate zero-time values were determined for each ionic condition.) Furthermore, none of t h e t r e a t m e n t s interfered with dissolution of polyphenylalanine in m-cresol nor with its subsequent passage t h r o u g h t h e filter. Acetates were always used for incubations, since Mg a÷, K ÷ a n d a v a r i e t y of other acetates are readily soluble in cresol, whereas chlorides are insoluble a n d m a y give b u l k y precipitates, causing self.absorption.

(vi) Reproducibility and accuracy I n each of two experiments, 30 identical samples were precipitated with cresol a n d filtered under s t a n d a r d conditions. S t a n d a r d deviations from m e a n values were 4.5% a n d 2.5% in respective expe "rnnents. E r r o r was reduced in t h e present work b y routine use o f duplicates or triph'cates. Since the e x t e n t o f reaction is calculated b y difference from u n t r e a t e d samples, small release values are subject to relatively large error. The m e t h o d is therefore unsuitable for measurement of initial rates. However, extents of reaction in t h e range S0 to 70% can be estimated with relative accuracy.

(vii) Gorrelation with other assays The cresol assay is based on the observation t h a t chemically synthesized polyphenylalanine (Sigma) is readily soluble in cresol, whereas t R N A a n d p o l y p h e n y l a l a n y l - t R N A are precipitated. The v a l i d i t y of t h e assay has been confirmed b y correlation w i t h two independent methods for determining the extent of reaction. Table 1 shows results of parallel determinations on several different reaction mixtures b y t h e SDS-sucrose gradient procedure (Gilbert, 1963; T r a u t & Monte, 1964) a n d b y t h e cresol assay. Estimates for percentage reaction agree to within experimental error. The puromycin reaction can also be followed from u p t a k e of puromycin into t h e released peptides (Allen & Zamecnik, 1962; Nathans, 1964; Smith, T r a u t , B l a c k b u r n & Monro, 1965). Using a 32P-labelled analogue o f puromycin (puromyein 5"-fl-cyanoethylphosphate, TXBT.~. 1

Gorrelation of cresol and sodium dodecyl sulIahate Release of polyphenylalanine (%) Assay method

(a)

(b)

(c)

(d)

(e)

Cresol

24

29

36

39

55

SDS

26

30

32

41

46

Compilation of representative data from experiments using several preparations of charged ribosomes and varied conditions of incubation with puromycin. After incubation, samples were divided, part being used for cresol assay and part for SDS assay. Zero-time samples without puromycin were treated in a similar way to determine amount of polyphenylalanyl-tR~qA initially present. The cresol assay was as described in text (Materials and Methods, (d) (ii) and (Hi)), and the SDS assay was by the procedure of Traut & Monte (1964).

THE POLYPHENYLALANINE

SYSTEM

337

P C E P ) (Smlth et aL, 1965) u p t a k e of label into trichloroacetic acid-preclpltable m a t e r i a l was determined after incubation w i t h [SH]polyphenylalanme-charged ribosomes (Monro, T r a u t & Blackburn, unpublished results). Q u a n t i t a t i v e correlation of P C E P - u p t a k e w i t h cresol assay values was n o t a t t e m p t e d for technical reasons, b u t several results (all those tested) obtained using the cresol assay were confirmed b y the P C E P assay. U p t a k e of s2p was dependent upon pre-charging the ribosomes with polyphenylalanine, a n d was inhibited b y pre-incubation of charged ribosomes with puromycin. Tt was observed with charged 50 s subunits as well as w i t h charged 70 s ribosomes, was s t i m u l a t e d a t high concentrations of NH4 + (c£ Maden & Monro, m a n u s c r i p t in preparation) a n d inhibited b y chloramphenicol (e£ T r a u t & 1Konro, 1964). The results (I) support t h e v a l i d i t y of the cresol assay a n d (2) indicate t h a t the polyphenylalanine released from t R N A in t h e present s y s t e m i s transferred to puromycin, as required for a model system for p e p t i d e b o n d formation.

3. Results (a) Non-involvement of 8upernatant factors and guanosine tril~hosphate Figures 1 and 2 show typical progress curves of the reaction between puromycin and salt-washed, polyphenylalanine-charged ribosomes in a solution containing only monovalent and divalent cations and buffer. The rapid initial rates clearly indicate that the reaction takes place readily under such minimal conditions.

II 40

(a) 30° C

A

(hi o°c 20

0

;;

01 3

A

A

;

! 3O

10

Time(mTn)

'6 1 ' 0

FzG. 1. Thne-oourses of puromycin reaction (70 s ribosomes). The reaction of salt-washed, [l~C]polyphenylalanine-charged ribosomes (see Materials and Methods (b)) with puromycln was assayed by the standard procedure (Materials and Methods,

(d) (ii)and (Hi)).

(a) Incubation at 30°C. ~0--0---,. Standard conditions; A , puromyc~u omitted. (b) Incubatio~ at 0°C. - - $ - - 0 - - , Standard conditions; ~O--O'~'s pre-iueubated 5 miu at 30°C immediately prior to assay at 0°C; A, puromycin omitted.

338

B. E. H. MADEN,

R. R. TRAUT

AND R. E. MONRO

Table 2 shows the effects of successive washes of polyphenylalanine-charged ribosomes with 0.5 ~-NH~C1 solution (in 0.01 M-Tris-HC1 buffer (pH 7.4) plus 0.01 Mmagnesium acetate). A single wash enhanced rather than lowered reactivity towards puromycin. There was no reduction in reactivity after two further washes, and little or no reduction even after a total of five washes. Ribosomes washed under similar conditions are dependent upon supernatant factors and GTP for polypeptide synthesis (Nishizuka & Lipmann, 1966). Our results therefore suggest that the puromycin reaction does not involve GTP or supernatant factors. !

!

I

I

!

|

4o

20

0 1 2

I

5

I

20

I0 Time (rain)

FIe. 2. Time-courses at 0°C and reduced puromycin concentration (70 s ribosomes). The reaction of [14C]polyphenylalanine-charged ribosomes with puromycin was assayed by the standard procedure (Materials and Methods, (d)(ii) and (iii)) at 0°C. - - $ - - $ - - , Staadard puromycin concentration (5 × 10 -4 ~); m e - - o - - , 2 × 10 -5 ~-puromycin.

TABLE 2

Effects of u~hing charged ribosomes on reactivity towards puromycln Release of polyphenylalanine (%) Exp. 1 Unwashed 3 times washed 5 times washed Exp. 2 Unwashed Once washed 3 times washed

1 min, 0°C 17 31 32

30 rain, 30°0 63(5) 67(6) 74(11)

1 min, 30°C 32 52 49

[14C1Polyphenylalanine-charged ribosomes were washed by centrifugation in 0.01 M-Tris buffer (pH 7.4), 0-01 M-magnesium acetate and 0-5 ~-17H4C1. Samples of the ribosomes were assayed for reaction with puromycin by the standard proced~lre (Materials and Methods, (d) (ii) and (iii)) at 0°C and 30°0. Figures in parentheses represent estimates for release after incubation without puromyein. Different ribosome preparations were used in the two experiments, and the concentration of ribosomes was 1 mg/ml, in exp. 1 and 0.2 mg/ml, in exp. 2.

(b) Ro~e of 50 ~ 8ubunit As previously observed (Traut & Monro, 1964), polyphenylalanine-charged 50 s subunits react with puromycin in the absence of 30 s subunits. Results in Figure 3 show that this reaction also occurs without added supernatant and GTP. In general,

THE POLYPHEI~YLALANII~E

SYSTEM

339

the reaction with 50 s ribosomes has characteristics slm~iar to the 70 s system (see sections (c) and (d), below; and Maden & Monro (manuscript in preparation) ). The reaction of charged 50 s subunits with puromycin is inhibited b y chloramphenicol (]~Ionro, unpublished results), as is the reaction with 70 s ribosomes (Traut & Monro, 1964). The occurrence of a reaction between puromyein and polyphenylalanyl-tRNA on 50 s subunits, together with the demonstration t h a t added supernantant factors are not required, suggests t h a t the puromyein reaction is catalysed b y the 50 s ribosome itself. i l

i

II

I

I

i i

II

i

i

'

Io'

l

6O

~

.

N

20

o

-1

-"

s

Js

II

~

3o~1 a Time (rain)

(a)

"'11

ao

(b)

FIG. 3. Time-courses of puromycin reaction with 50 s subunits. [14ClPolyphenylalanine-charged 50s ribosomal subunits were prepared as described under Materials and Methods (c), and assayed for reaction with puromycin under standard conditions (Materials and Methods (d) (ii) and (iil)) at 30°C. (a) Pre-ineubated 30 min at 80°C before addition of puromycin. During this time, 17% of the radioactive material was released. Percentage reaction in the graph is calculated on the basis of the cresol value after pre-incubation. (b) No pre-incubation. - - O - - O - - , Standard conditions; A, no puromycin. (c) Kinetics Kinetics of the reaction between salt-washed polyphenylatanine-eharged ribosomes and puromycin are highly heterogeneous. Typical progress curves are shown in Figures 1 and 2. At 30°C and 5 × 10 -4 M-puromycin, appro~ir-ately 50~/o of the polyphenyl. . . . alanine was released in less 1than one mlnuto (Fig. l(a)). An additional 3 0 0~ was released more slowly, and 2 0 ~ failed to react within 30 minutes. The time-course for the most rapidly reacting material could only be followed b y carrying out the reaction at 0°C and reduced puromycin concentration (2 × 10 -5 M) (Figs l(b) and 2). Kinetics with 50 s subunits arc qualitatively siml]ar, but both rate and extent of reaction are lower (Fig. 3). Neither the rapid nor the slow phases of the progress curves in Figures 1 to 3 can be fitted to exponential functions (Maden, 1966). However, the possibility t h a t a small, highly active fraction of the ribosomes reacts with exponential ]~netics could not readily be determined for technical reasons (lYr_a~rials and Methods, (d) (vi)). Some factors which have been found t o affect the reactivity of the ribosomes are as follows. (1) Ribosomes washed with 0.5 ~-NH4C1 (or KC1) are the most reactive (Table 2). T h e peptidyl transferase activity of uncharged ribosomes is also activated b y salt-

8t0

B.E.H.

MADEN, R. R. TRAUT AND R. E. MONRO

washing (Monro, 1967). High salt concentrations in the reaction m i n u t e stimulate the reaction without changing the heterogeneity of the ~{notics. Conversely, exposure, b y washing or dialysis, of charged ribosomes to media lacking monovalent cations inhibits the rate of subsequent reaction in the presence of 0.I M-ammonium acetate ( ~ d e n & 1~Ionro, manuscript in preparation). I t is possible that more reactive preparations of charged 50 s subunits might be obtained b y suitably modifying the ionic conditions used for their preparation. (2) A short pre-incubation a t 30°0 results in slight stimulation (5 to 10~/o more release) of the early phase of the reaction at 0°C (Fig. 1). (3) The reaction does not proceed at maximum rate at the p H value employed in the present experiments (pH 7.4, as for polyphenylalanine synthesis). However, the ~inetics at optimal p H value (above p H 8.5), though more rapid, were no less heterogeneous t h a n at p H 7.4 (Maden & Monro, manuscript in preparation). As a result, both of the heterogeneous kinetics and of the technical diP~culty of following the early progress of the reaction, measurements of initial rate have not regularly been carried out. Effects of various conditions on the reaction have been described in terms of percentage release values after shor~ and longincubationperiods (usually 1 rain at 0 ° or 30°0, or 30 rain at 3000) in order to show up changes in rate or extensive inactivation. (d) Puromyci~ con~ntratio~ Figure 4 shows the effect of puromycin concentration on the reaction. I n the experiment recorded, the 70 s ribosomes were incubated with puromycin for one minute at 0°C and the 50 s subunits for four minutes at 30°0. The 70 s preparation was more reactive, but the curves are qualitatively s{milar. Puromycin concentrations as low as 10-6 M brought about release of polyphenylalanine, and the rate of release

40 II '

'

i

g: 2{

......I, 10-6

I I 10S 10-4 Puromycln concentrotion (~0

!

10-3

FIG. 4.~Effect of puromyc~ coneenf~at,ion.

The reaction of [140]polyphenylalanine-charged ribosomes with puromycin was assayed by the standard procedure (Materials and Methods, (d)(ii) and (iii)) with puromyein concentration varied as indicated, i O - - O - - - , 70 s ribosomes, inqubated 1 min at 0°C; I C ) - - O - - , 50 S subunits, incubated 4 mln at 80°C. Blank release which occurred in ~ho absence of puromycin has been subtracted.

T H E P O L Y P H E N Y L A L A N I N E SYSTEM

341

continued to increase up to 5 ×10 -~ M. A saturating puromycin concentration was not defined, b u t in other experiments a furtber 7 to 9 % increase in the percentage release occurred on increasing the puromyein concentration from 5 X 1 0 - 4 ~ t o 5 × 10 -3 M. I n most experiments we have used a concentration of 5 × 10 -4 ~.

(e) Effects of urea, sodium dodecyl ~l~hate, ex~oeure to high temperature and formal. dehyde The puromycin reaction was completely inhlbited b y 4M-urea or 0.5% SDS (Table 3), b y incubating the ribosomes for five minutes a t 70°C (Fig. 5) or b y prior exposure of the ribosomes to formaldehyde (2% for 16 hr a t 0°C) followed b y removal of the unreacted formaldehyde b y dialysis (Table 4). Treatment for one hour at 0°C with 1 to 5% formaldehyde led to partial inactivation. Controls show t h a t the cresol assay was not affected b y urea or SDS. The findings indicate t h a t native ribosomes are required for catalysis of the reaction. T~BLE 3

Effec2z of urea and sodium dodecyl ~ulphate o~ the ~uromycin reaction Release of polyAddition

Exp. 1 (a None (b) 4 M-urea, immediately before cresol (C) 4 M-urea (d) 4 M-urea, then dialysed

EXP. 2 (a) None (b) 2% SDS, immediately before cresol (c) 0.5% SDS, pro.inc. 1 mln, 30°G (d) 0"5% SDS, pro-inc. 10 rain, 30°C (e) 2% SDS, pro-inc. 1 min, 30°0

phenylal~nlne (%) I min 30 rain

43

73(13) 80

4 23

13(10) 45(18)

73 72 26 10 9(6)

Assay of the reaction of polyphenylalanine-eharged ribosomes with puromycin was carried out b y the standard procedure (Materials and Methods, (d) (ii) and (iii)) with the additional treatments shown. Incubation was at -30aC. I n treatment (d) (exp. 1), 4 xc-urea was added to a portion of reaction m~Yture (without puromyein) anc~the r n ~ u r e was dialysed for 4 hr against 0.01 MTris buffer (pH 7.4) and 0.01 ~-magnesium acetate. The preparation was then assayed for reaction with puromycin under standard conditions. Figures in parentheses represent estimates for release in absence of puromycin.

Inhibition produced b y 4 ~-urea could be partially reversed on removal of the urea b y dialysis. An appreciable time was required for urea or SDS to produce complete inhibition: 30 to 60 seconds for 4 ~-urea (data not shown), more t h a n one minute for 0.5% SI)S, and less t h a n one m~uute for 2 % SDS. Incubation o f t b e charged ribosomes in the range 30 to 60°C led to a small but reproducible increase of reactivity (Fig. 5). I t was of interest t h a t ribosomes were inactivated as regards protein synthesis over a temperature range approTimately 10 dog. C lower t h a n the range over which f~e puromycin reaction was inhibited (Fig. 5). Whether this signlf~es greater thermal

342

B. E . H. M A D E N , R. R. T R A U T A N D R. E . M O N R O

lability of part of the ribosome connected with some function other than peptide bond formation, or merely partial protection of the ribosome against the effects of high temperature by the binding of peptidyl-tRNA, was not determined. T~LE 4

Effect of pre.treatment with formaldehyde on the puromycin reaction • Release of poly-

phenylalanine (%) I min

30min

Exp. I: (1 hr at 0°C) No HCHO 1% HCHO 2% HCHO 5% HCHO

41 25 24 10

75(15) 63(13) 59(15) 46(16)

Exp. 2:(16 hr at 0°C) No HCHO 2% HCHO

45 2

71(3) 20(0)

[14CJPolyphenylalanine-charged ribosomes (0.4 mg]ml.) in 0.05 ~t.triethauolamine buffer (pH 7.4) and 0.01 ~-magnesium acetate were treated with formaldehyde for the times and at the concentrations indicated in the Table. After removal of free formaldehyde by dialysis (4 hr) against 0.01 ~-Tris buffer (pH 7.4) and 0.01 ~t-magnesium acetate, the ribosomes were assayed for reaction with puromyein by the standard procedure (Materials and Methods, (d)(ii) and (iii)). Incubation was at 30°C. Figures in parentheses represent release values after incubation without puromycin.

4. Discussion

(a) Minimal requirements for the Turomycin reaction The experiments described above suggest that supernatant factors and GTP are not directly involved in the reaction of puromyein with polyphenylalanine-charged ribosomes. Evidence from inhibitor studies supports this conclusion. The reaction is not inhibited by sulphydryl group reagents (Traut & Monro, 1964; Monte et al., 1967), which inactivate the "G" and "Tu" supernatant factors, nor by guanylyl-methylenediphosphonate (Monro et al., 1967), a specific inhibitor of the GTP reaction in protein synthesis (Hershey & Monte, 1966). The polyphenylalanine studies do not, however, eliminate the possiblity that a molecule of a supernatant enzyme is firmly and specifically bound to the peptidyltRNA-50 s subunit complex, in a state which is resistant to inhlbitors. This possibility has now been eliminated by studies with uncharged ribosomes. Salt-washed ribosomes eatalyse the reaction of puromycin with polylysyl-tRNA (in the presence of poly A) (RychlilL 1966), and also with formylmethionyl-tRNAin presence of suitable template to give formylmethionyl-puromycin (Bretseher & Mareker, 1966; Zamir, Leder & F,lson, 1966). More recently, isolated 50 s subunits have been shown to catalyse the reaction of puromycin with a formylmethionyl-hexanueieotide, CAACCA-Met-F (the fragment reaction; Monte, 1967). In this system, similar salt-washing and inhibitor studies to

THE

POLYPHENYLALANINE

--41

,

.

3C

,

,

SYSTEM

343

,

O

1~ ~

60

2c

4o

•~ I0

20

o

0

40 50 60 Pre-incubation temperature (~)

70

~'~Q. 5. ~,ffect of pre-treating ribosomes at elevated temperature on phenylalanine incorporation and on the puromycin reaction. - - 0 - - 0 - - , Unwashed E. co// B ribosomes (1 mg/ml,) in 0.01 M-Tris buffer (pH 7.4) and magnesium acetate were incubated for 5 rnln at the temperatures shown, and then assayed for incorporation of [z~C]phenylalanino into the hot triehloroacetic acid-insoluble fraction. Conditions for incorporation were those described under Materials and Methods (b), except that ribosome concentration was 0-5 mg/ml.; incubation volumes were 0.2 ml., and reactions were terminated after I0 rain by addition of 5% trichloroacotic acid. ~ O - - O - - , Unwashed, [z4C]polyphenylalanine-eharged E. coli B ribosomes (1 mg/ml.) in 0.01 M-Tris buffer (pH 7.4) and magnesium acetate were incubated for 5 min at the temperatures shown and then assayed for reaction with puromycin by the standard procedure (Materials and Methods, (d) (ii) and (iii)). Incubations were for 30 min at 30°C, at a ribosome concentration of 0.5 mg/ml. Separate zero time determinations were carried out on each sample, and indicated that there was little release of polyphenylalanine from tRNA during the pro-incubations at elevated temperatures (about 15~zoloss after 5 mln at 60°0 or 67°C, less than 4% loss after pre-ineubation at 50°0). Essentially identical results were obtained in another experiment, in which [SH]polyphenylalanine-charged ribosomes were used for assay of both reactivity towards puromycin and synthesis of polyphenyla]anine (using 14C-labelled phenylalanine).

those used in the polyphenylalanlne s y s t e m were carried out, a n d t h e possibility o f catalysis b y a specifically b o u n d a n d p r o t e c t e d s u p c r n a t a n t e n z y m e was e|imlnated. / • B o t h the f r a g m e n t a n d t h e p o l y p h e n y l a l a m n e systems suffer f r o m disadvantages: namely, dependence of the f r a g m e n t reaction u p o n alcohol (Monro & 1VIarcker, 1967), a n d t h e possibility o f a b o u n d s u p e r n a t a n t protein in the polyphenylalanine system. However, results f r o m t h e two systems are c o m p l e m e n t a r y , a n d t o g e t h e r provide v e r y strong evidence t h a t t h e 50 s subunit, itself, is responsible for catalysis o f t h e p u r o m y c i n reaction. The recent report t h a t 50 s subunits in addition to 30 s subunite a n d A p U p G are required for the reaction of f o r m y l m c t h i o n y l - t R N A with p u r o m y c i n (F[fllc, Miller, I w a s a k i & W a h b a , 1967) are consistent with this conclusion. (b) Relation of puromycin reaz~ion to normal peptide bond formation T h e following lines o f evidence, considered more fully elsewhere (Monro etal., 1967), indicate t h a t t h e p u r o m y c i n reaction takes place b y the same m e c h a n i s m as peptide b o n d f o r m a t i o n in protein synthesis.

344

B.E.H.

M A D E N , R. R . T R A U T A N D R . E. M O N R O

(i) Chemical slmilarlties of the substrates and products of the reaction and specificity towards substrates. The T,-phenylalanine analogue of puromycin is active, whereas the D-analogue is inactive. Analogues with the amino acid on the 2' or 5' rather than the 3' position of the amino-nucleoside are inactive (Nathans & Neidle, 1963). (See Monro, Cerna & Marcker (manuscript in preparation) for further consideration of substrate specificity.) (ii) Dependence upon (native) ribosomes. (iii) Dependence upon monovalent and divalent cations (see also Maden & Monte, manuscript in preparation and references therein). (iv) Action of specific ~nh~bitors of protein synthesis (see also Monte & Vazquez, 1967 and references therein). The results from the polyphenylalanine and fragment reactions therefore signify that normal peptide bond formation is catalysed by the 50 s ribosomal subunit. The recent demonstration that salt.washed ribosomes can catalyse the transfer of polylysine from tRNA to lysyl-tRNA (with formation of a single peptide bond) (Gottesman, 1967) provides independent evidence that peptide bond formation in protein synthesis is catalysed by the ribosome itself, although in this case the role of the 50 s subunit was not distinguished. (e) Mechanism and enzymi~ nature of the rear, ion Non-involvement of GTP in the puromycin reaction suggests that peptide bond formation proceeds by a simple group-transfer mechanism involving nucleophillc attack of the ~-NH 2 group of puromycin (or amlnoacyl-tRNA) on the carboxyl ]inl~age of peptidyl-tRNA. Evidence in the previous section suggests that the 50 s ribosome functions catalytically in the reaction. This conclusion is supported by the observation that the reaction proceeds rapidly at low puromycin concentration (10-6 to 10-4 ~), in contrast to the (presumably) chemically related but non-enzymic reaction of hydroxylamine with polyphenylalanyl-tRNA (Gilbert, 1963). It is probable that a Michaelis type complex is formed between puromycin and a site on the 50 s subunit, thus orienting the attacking ~-NH 2 group of puromycin with respect to the acyl llnl~age of poptidyl-tRNA (and, possibly, a functional group at the catalytic centre which promotes the reaction). For technical reasons (see Materials and Methods and Kinetics sections) we did not attempt to determine K m for puromycin. It appears reasonable to identify catalysis of peptide bond formation by 50 s subunits with classical enzyme reactions, and to suppose that the catalytic centre is on a protein molecule integrated into the ribosome structure (Monro eta/., 1967, p. 202-3). Non-involvement of GTP, suggests that the enzyme responsible falls into the category of tran~ferases (Report of the Commission on Enzymes, 1965). Peptide synthetase is a misnomer, since synthetase reactions, by definition, involve nucleoside triphosphates. We have proposed peptidyl transferase as a suitable operational name (Monro et al., 1967, p. 187). GTP and supernatant factors are presumably involved at steps other than poptidyl transfer in the cycle of events which occurs during the addition of each amino acid to the growing peptide chain. The observation that the reaction of polyphenylalaninecharged ribosomes with puromycin is stimulated to a variable extent by GTP and supernatant led to the postulate that GTP and a superuatant factor are involved in a step prior to peptidyl transfer, and possibly constitute a translocaze enzyme system which shifts peptidyl-tRNA from one site to another site on the ribosome (Traut &

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Monro, 1964). This aspect of the polyphenylalanine system has not been examined further, a n d more defined systems for such studies are now available (Hershey & Thatch, 1967; Lucas-Lenard & Lipmann, 1967; Allende & Weissbach, 1967; Anderson, Bretscher, Clark & Marcker, 1967; Leder & Nau, 1967). We are gratefill to Dr S. Brenner and Dr F. H. C. Crick for s~imulating discussions during the course of this work. We also thank the Microbiological Research Station, Porton, for preparation of the E. coli cells. One of us (B. E. H. M.) gra~efully acknowledges the receipt of a Medical Research Council scholarship for training in research methods, and during part of the work, he was in the Department of Radiotherapcu~ies, University of Cambridge. REFERENCES Allen, D. W. & Zamecnik, P. C. (1962). Biochim. biophya. Acta, 47, 865. Allende, J. E. & Weissbach, H. (1967). Bioch~m. Biophys. Rez. Comm. 28, 82. Anderson, ft. ft., Bretscher, M. S., Clark, B. F. C. & Marcker, K. A. (1967). Nature, 215, 490. Bretscher, M. S. & Marcker, K. A. (1966). Nature, 211, 380. Cammack, V. A. & Wade, H. E. (1965). Bioch6m. J . 96, 621. Gilbert, W. (1963). J . MoL Biol. 6, 389. Gottesman, M. (1967). J . Biol. Chem. 242, no. 23, p. 5564. Hershey, ft. W. B. & lYlonro, R. E. (1966). J . Mol. Biol. 18, 68. Hershey, J. W. B. & Thatch, R. E. (1967). Proc. Nat. Acad. ~ei., Wash. 57, 759. Hille, M. B., Miller, M. J., Iwasaki, K. & Wahba, A. J. (1967). Proe. Nat. Aead.Sci., Wash. 58, 1652. Leder, P. & Nau, M. M. (1967). Proc. Nat. Acad. Sci., Wash. 58, 774. Lucas-Lenard, ft. & Lipmann, F. (1967). Proc. Nat. Acad. ~ci., Wash. 57, 1050. Madcn, B. E. H. (1966). Ph.D. Thesis, Cambridge University. Matthaei, ft. H. & Nirenberg, M. W. (1961). Proc. Nat. Acad. Sci., Wash. 47, 1580. Monro, R. E. (1967). J . MoL Biol. 26, 147. Monro, R. E., Maden, B. E. H. & Traut, R. R. (1967). Syrup. Fed. European Biochem. Soc. (April, 1966), ed. by I). Shugar, p. 179. London: Academic Press. Monro, R. E. & Marcker, K. A. (1967). J . Mol. Biol. 25, 347. Monro, R. E. & Yazquez, D. (1967). J . Mol. Biol. 28, 161. Nathans, D. (1964). Proc. Hat. Acad. Sci., Wash. 51, 585. Nathans, D. & Neidle, A. (1963). 17ature, 197, 1076. Nishizuka, Y. & Lipmann, F. (1966). Proe. Nat. Acad./Jci., Wash. 55, 212. Rychlik, I. (1966). Biochim. biophys. Acta, 114, 425. Smith, J. D., Traut, R. R., Blackburn, G. M. & Monxo, R. E. (1965). J . Mot. Biol. 18, 617. Traut, R. R. & Monro, R. E. (1964). J . Mol. Biol. 10, 63. Zam~r, A., Leder, P. & Elson, D~ (1966). iProc. Nat. Acad. ~ci., Wash. 56, 1795.