ARCHIVES

OF BIOCHEMISTRY

Masking

AND

169, 622-626

(1975)

of Peptidyl Transferase VAGN

Department

BIOPHYSICS

of Biology,

R. LEICK’ Massachusetts

AND

Activity

ROBERT

Institute

in Polyribosomes

F. SANTERRE

of Technology,

Cambridge,

Massachusetts

02139

AND

RAYMOND The Biological

Laboratories,

KAEMPFER

Harvard Received

University, December

Cambridge,

Massachusetts

02138

24, 1974

The peptidyl transferase activity of polysomes from Escherichia coli, rabbit reticulocytes and chick embryos, assayed in the fragment reaction, is 3- to lo-fold lower than the corresponding activity of single ribosomes. The polysomal peptidyl transferase activity is restored in full under conditions of in vitro protein synthesis that result in conversion of polysomes to single ribosomes. Thus, the peptidyl transferase center is masked in translating ribosomes. Unmasking of peptidyl transferase, however, does not require the release of ribosomes from messenger RNA: it is also seen upon treatment of polysomes with puromycin, under conditions in which polysomes remain intact. Apparently, release of nascent polypeptide chains is sufficient to allow access of formylmethionyl hexanucleotide substrate to the peptidyl transferase site.

(Escherichia coli) and two eukaryotic sources (rabbit reticulocyte and chick embryo) are shown to possess low measurable PTase activity whereas their corresponding single ribosomes have maximal activity. The PTase activity of polysomal ribosomes becomes fully detectable when they are allowed to accumulate as single ribosomes in the course of cell-free protein synthesis. These findings suggest that in translating ribosomes the presence of peptidyltRNA blocks access of the hexanucleotide substrate used to measure PTase, and that the access is restored when ribosomes are released from messenger RNA upon termination of protein synthesis. However, we find that complete unmasking of PTase activity does not require the release of ribosomes from messenger RNA: intact, purom ycin- treated polysomes display maximal PTase activity.

Peptide bond formation in protein synthesis is catalyzed by peptidyl transferase (PTase),’ an enzyme, or enzyme center, that is an integral part of the large ribosomal subunit (1, 2). This enzyme catalyzes the transfer of the nascent polypeptide chain from peptidyl transfer RNA to the a-amino group of aminoacyl-tRNA, or to the a-amino group of puromycin (3, 4). A simple assay for PTase, the so-called fragment reaction (5-71, involves the use of a hexanucleotide fragment, generated from N-formylmethionyl-tRNA by T, RNase digestion, as donor of N-formylmethionine to puromycin. We report here that ribosomes contained in polysomes differ from single ribosomes in their PTase activity as detected by the fragment reaction assay. Polysomal ribosomes from one prokaryotic organism I Present address: Biokemisk Institut B, Panum Instituttet, Blegdamsvej 3, DK-2206 Kebenhavn N, Denmark. 2Abbreviations used: PTase, peptidyl transferase; and PM, puromycin.

MATERIALS

622 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND

METHODS

Preparation of E. coli (strain DlO or MREGOO) polysome lysates and in vitro protein synthesis (S),

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OF PEPTIDYL

preparation of rabbit reticulocyte polysome lysates and cell-free protein synthesis (9), and preparation of polysome extracts from chick embryos (g-day-old White Leghorn) (10) were as described. Polysomes and single ribosomes were prepared by collecting the appropriate fractions from sucrose gradients and concentrating them by centrifugation for 3 h at 321,000g in tubes containing a 75% sucrose cushion. Salt-washed E. co/i DlO ribosomes were prepared as described (11). N-Formyl [%]methionyl hexanucleotide was prepared from purified N-formylmethionyltRNA, (fMet-tRNA) and 35S-labeled methionine (19 Ci/mmol) (Amersham) as described (12). The hexanucleotide fragment was generated by RNase T, (13). The fragment reaction assay was as described (14). Buffer B: 0.02 M Tris (pH 7.41, 0.005 M MgCl,, 0.1 M KCl. Buffer C: 0.01 M Tris (pH 7.4), 0.01 M Mg-acetate, 0.05 M KCI. RESULTS

The PTase activity of ribosomes isolated from a polysome lysate of exponentially growing E. coli is shown in Fig. 1. The activity of such ribosomes (curve b) is about tenfold lower than that of saltwashed single ribosomes (curve f). Preincu-

0

20

40

60

MINUTES

FIG. 1. Peptidyl transferase activity of ribosomes in an E. coli polysome lysate before and after cell-free protein synthesis. An E. coli MREGOO polysome lysate was incubated at 37°C under conditions of cell-free protein synthesis, for 0 min (b, c) and 20 min (d, e). After cooling to O”C, the ribosomes were isolated by centrifugation and resuspended in 100 ~1 of buffer C. The ribosomes of each sample were assayed in the fragment reaction before (b, d) and after (c, e) incubation at 37°C for 10 min in buffer C. Saltwashed E. coli DIO single ribosomes were assayed in the complete assay (f) and in the absence of puromytin (a). Arrow on ordinate indicates input radioactivity.

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FIG. 2. Sedimentation distribution of ribosomes in an E. coli MREGOO polysome lysate. The lysate was centrifuged for 3.4 h at 41,000 rpm and 5°C in an International SB283 rotor through a 13.3-19.6s (w/v) exponential sucrose gradient (11 ml) supported by a 1.5ml cushion of 75% sucrose in buffer C, and the A,,,, of the gradient was monitored through an ISCO flow cell. The polysomes and single ribosomes were pooled as indicated and concentrated by ultracentrifugation.

bation of the lysate at 37°C in the presence of 0.05 M KC1 does not lead to an appreciable increase in the PTase activity (curve c). However, when a polysome lysate is first incubated at 37°C under conditions for protein synthesis that favor the accumulation of single ribosomes at the expense of polysomes (15), and ribosomes are then isolated, full restoration of their PTase activity is observed (curve d). Once a lysate has been preincubated, incubation of the isolated ribosomes at 37°C in the presence of 0.05 M KC1 does not increase their activity further (curve e). These results suggest that the increase in detectable PTase activity is due to an increase in the proportion of single ribosomes. To measure the PTase activity of polysomes and single ribosomes directly, the two classesof particles were isolated from sucrose gradients as shown in Fig. 2, and then assayed in the fragment reaction. As seen in Fig. 3, polysomes (curve b) indeed display much less activity than single ribosomes (curve d). Incubation of polysomes at 37°C in the presence of 0.05 M KC1 fails to cause a significant stimulation of the activity (curve c), indicating that the low activity of polysomes is not due to a reversible inactivation (16). To determine if the PTase activity of

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LEICK, I

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SANTERRE

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E. COLI

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MINUTES FIG. 3. Peptidyl transferase activity of isolated E. coli MREGOO single ribosomes and polysomes. Single ribosomes and polysomes were isolated as described in Fig. 2. Isolated polysomes (b,c) or single ribosomes (d,e) were assayed in the fragment reaction immediately (b,d) or after preincubation for 15 min at 37’C in buffer C (c,e). The ribosome concentration during preincubation was 2 mg/ml. Curve (a): polysomes assayed in the absence of puromycin. Arrow on ordinate indicates input radioactivity.

AND

KAEMPFER

concentration (0.1 M KCl). As seen in Table I, column 3, such treatment results in an almost complete restoration of the PTase activity of polysomes to the level of single ribosomes. Some stimulation of the polysomal activity also occurs upon incubation at 37°C in the absence of puromytin, but it is less pronounced (Table I, column 2). Essentially identical results are obtained when the preincubation of polysomes is conducted at high salt concentration (0.8 M KCl; Table I, columns 4-6). Figure 5 shows the effect of preincubation and puromycin treatment on the sedimentation distribution of chick embryo polysomes. As expected, during preincubation of polysomes at high salt concentration (d-f) the restoration of PTase activity is concomitant with the conversion of polysomes to single ribosomes and subunits. By contrast, the extent of restoration of PTase activity obtained by preincubation at low salt concentration (see Table I) is not matched by a corresponding change in the

RETICULOCYTE I

eukaryotic ribosomes behaves similarly, polysomes and single ribosomes were isolated from a rabbit reticulocyte lysate before and after cell-free protein synthesis in the absence of added hemin, a treatment known to convert almost all polysomes to single ribosomes (17, 18). As seen in Fig. 4, reticulocyte polysomes (curve b) possess almost no detectable PTase activity compared to single ribosomes (curve d). A slight stimulation is observed when the polysomes are preincubated at 37°C (curve c), but they do not attain the level of single ribosomes (curve e). The activity of reticulocyte single ribosomes (curves d, e) is comparable to that of E. coli single ribosomes (curve f). As in the case of E. coli and rabbit reticulocytes, single ribosomes from chick embryos also display considerably greater PTase activity than the corresponding polysomes (Table I, column 1). Using these polysomes, we examined the effect of preincubation with puromycin at low salt

MINUTES

FIG. 4. Peptidyl transferase activity of rabbit reticulocyte single ribosomes and polysomes. Polysomes (b,c) and single ribosomes (d,e) were isolated and assayed in the fragment reaction. Assays were carried out immediately (b,d) or after preincubation for 15 min at 37°C in buffer C (c,e). For comparison, equal numbers of E. coli salt-washed single ribosomes were assayed in the presence (f) and absence (a) of puromytin. Arrow on ordinate indicates input radioactivity.

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OF PEPTIDYL TABLE

PEPTIDYL

TRANSFERASE

ACTIVITV

Preincubation

Single

I

EMBRYO

SINGLE

RIBOSOMES

0.1 M KC1 (cpm)

ribosomes

Polysomes a Isolated polysomes puromycin (2 mM) and incubated for 20 min puromycin formed. An cpm. A blank value of

OF CHICK

625

TRANSFERASE

0°C

37°C

1751

1800

540

958

AND

POLYSOMES”

0.8 M KC1 (cpm) 37°C + Puromycin

0°C

37°C

37°C + Puromycin

1836

1709

1545

1953

1664

581

1108

1518

or single ribosomes in buffer B were preincubated for 30 min at 0 or 37°C with KC1 present as indicated. The samples, each containing 0.1 mg of ribosomes, were then in the fragment reaction assay. The numbers represent cpm of N-formylmethionyl equal number of salt-washed E. cob DlO single ribosomes in buffer C gave a value of 2310 326 cpm (single ribosomes assayed in the absence of puromycin) was subtracted.

_ + -+--~-~

-e

-

OEM 37’C

KCI ;

f OEIM KCI 37’C:PM

& 06

FIG. 5. Sedimentation distributions of chick embryo with puromycin (PM). Isolated chick embryo polysomes indicated KC1 concentration for 30 min at 0°C (a,d) or included in samples c and f. After fixation for 30 min with of 30% formaldehyde in 0.5 M triethanolamineeHC1 (pH centrifuged for 1.5 h at 41,000 rpm and 4°C in a Spinco sucrose gradient. l&40% (w/v), in 0.02 M triethanolamine-HCl KCl.

sedimentation profile of the polysomes (Fig. 5a-c). In particular, even though treatment with puromycin at 0.1 M KC1 leads to an almost complete restoration of PTase activity, it does not significantly affect the proportion of ribosomes sedimenting as polysomes. The values shown in Table I represent the specific activities of PTase in the ribosome preparations under study. Thus, upon treatment with

polysomes before and after treatment were preincubated in buffer B at the 37°C (b,c,e,f). Puromycin (2 mM) was formaldehyde [incubation with l/5 vol 7.4) 1. 5 A,,, units of each sample were SW41 rotor through an II-ml linear (pH 7.4), 0.005 M MgCI,, 0.1 M

puromycin the specific activity of the polysome preparation increases more than threefold and becomes equal to that of single ribosomes. The slight increase in single ribosomes and 60s subunits seen in Fig. 5c cannot account for the observed increase in specific activity. These results show that conversion of polysomes to single ribosomes is not needed for restoration of PTase activity.

626

LEICK,

SANTERRE

DISCUSSION

We show here that polysomal ribosomes of both prokaryotic and eukaryotic origin possess a 3- to lo-fold lower level of PTase activity than single ribosomes, as assayed in the fragment reaction. This low activity is the result of a masking of the PTase center in polysomal ribosomes, for the PTase activity of E. coli and rabbit reticulocyte polysomes becomes again fully detectable unde conditions of cell-free protein synthesis that lead to the accumulation of single ribosomes at the expense of polysomes. Although we observe that conversion of polysomes to single ribosomes leads to the unmasking of PTase activity, release of ribosomes from messenger RNA is not required. Using chick embryo polysomes, we show that PTase activity can be restored almost fully by preincubation with puromycin at low salt concentration (0.1 M KCl) even though such treatment does not lead to an appreciable release of ribosomes from polysomes (Table I; Fig. 5). The PTase activity of rat skeletal muscle ribosomes can be stimulated by preincubation with puromycin at high salt concentration, 0.88 M KC1 (19), but as shown by Blobel and Sabatini (20), that condition leads to dissociation of virtually all polysomes into ribosomal subunits and to the release of nascent polypeptide chains as well as messenger RNA. Our finding is that release of nascent polypeptide chains alone is sufficient to restore PTase activity. Since puromycin in the A-site of polysomes can serve as an acceptor of the nascent polypeptide chain, leading to the formation and release of peptidyl puromytin, it is most likely that the second substrate in the fragment reaction, the N-formylmethionyl hexanucleotide, is unable to enter the P-site of polysomal ribosomes when that site is occupied by nascent

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KAEMPFER

peptidyl-tRNA, but is able to do so upon release of the nascent polypeptide chain. ACKNOWLEDGMENTS We thank Dr. Alexander Rich for his interest and support. Aided by grants P-569 from the American Cancer Society and GM-19,333 from the USPHS to R.K., and by grants from the NSF and USPHS to A. Rich. V.R.L. was supported by a longterm EMBO fellowship; R.F.S. was supported by a postdoctoral fellowship from the USPHS. REFERENCES 1. MONRO, R. (1967) J. Mol. Biol. 26, 147. 2. MADEN, B., AND MONRO. R. (1968) Eur. J. Biothem. 6, 309. M., AND DE LAHARA, G. (1959) Proc. 3. YARMOLINSKY, Nat. Acad. Sci. USA 45, 1721. 4. ALLEN, D., AND ZAMECNIK, P. (1962) Biochim.

Biophys. Acta 55, 865. 5. MONRO, R., AND MARCKER. K. (1967) J. Mol. Hiol. 25, 347. R., CERNA, J., AND MARCKER, K. (1968) 6. MONRO, Proc. Nat. Acad. Sci. USA 61, 1042. 7. CELMA. M., MONRO. R., AND VAZQUEZ. D. (1970)

Fed. Eur. Biochem. Sot. Lett. 6, 273. 8. KAEMPFER, R. 11968) Proc. Nat. Acad. Sci. USA 61, 106. W. 9. ADAMSON, S., HERBERT, E., AND GODSCHA~X, (1968) Arch. Biochem. Biophys. 125, 671. 10. HEYWOOD, S., DOWBEN, R., AND RICH, A. (1967) Proc. Nat. Acad. Sci. USA 57, 1002. 11. ANDERSON, J., BRETSCHER, M., CLARK, B., AND MARCKER, K. (1967) Nature (London) 215,490. 12. HERSHEY, J., AND THACH, R. (1967) Proc. Nat. Acad. Sci. USA 57, 759. 13. MARCKER, K. (1965) J. Mol. Biol. 14, 63. 14. FAHNESTOCK, S., NEUMANN. H., SHASHOUA, V., AND RICH, A. (1970) Biochemistry 9, 2477. 15. KAEMPFER, R. (1970) Nature (London) 228, 534. 16. MISKIN, R., ZAMIR, A., AND ELSON, D. (1970) J.

Mol. Biol. 54, 355. 17. ZUCKER,

W., AND SCHLLMAN,

H. (1968)

Proc.

Nat.

J. (1972)

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I. (1970)

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Acad. Sci. USA 59, 582. 18. KAEMPFER,

R., AND KAUFMAN,

Acad. Sci. USA 69, 3317. 19. STIREWALT,

W.,

AND WOOL,

Biochen. Sot. Lett. 10, 38. 20. BLOBEL,

G.,

AND SABATINI,

Acad. Sci. USA 68, 390.

D. (1971)

Proc.

Nat.