ANALYTICAL BIOCHEMISTRY 93, 238-243 (1979)

A Rapid and Sensitive Assay for the Detection of Eukaryotic Ribosome Dissociation Factors DAVID W. RUSSELL AND LINDA L. SPREMULLI

Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, 27514 Received A u g u s t 4, 1978 A rapid and sensitive a s s a y has been developed for the factor-dependent dissociation o f eukaryotic ribosomes. This a s s a y takes advantage of the observation that initiation factor elF-2 will bind Met-tRNAf met to 40 S subunits but not to 80 S ribosomes. Incubation o f wheat germ r i b o s o m e s at 1 mM Mg 2÷ results in their dissociation into 40 S and 60 S subunits. T h e s e subunits s p o n t a n e o u s l y reassociate when the Mg 2+ concentration is raised to 4 mM. However, if the incubation at 1 mM Mg z+ is carried out in the presence of an extract containing a ribosome dissociation factor, a certain portion o f the subunits will fail to reassociate when the Mg 2+ concentration is raised to 4 mM. T h e 40 S subunits remaining due to the presence o f the dissociation factor can bind [35S]Met-tRNAfmet in the p r e s e n c e of wheat germ elF-2. T h e [85S]Met-tRNAf met b o u n d to the 40 S subunits is readily detected by its retention on a Millipore filter.

The initiation of protein biosynthesis in eukaryotic cells begins with the assembly of an initiation complex containing MettRNAfmet, GTP 1 and mRNA on the 40 S ribosomal subunit. Under physiological concentrations of magnesium (4-5 mM) the 40 S and 60 S ribosomal subunits spontaneously reassociate to form 80 S ribosomes. The cell must, therefore, have some mechanism to ensure an adequate supply of ribosomal subunits for initiation-complex formation. A number of laboratories have reported ribosome dissociation factors in eukaryotic cells including Xenopus (1), yeast (2), rat liver (3), rabbit reticulocytes (4-7), ascites cells (8), and bean leaves (9). Recent studies in our laboratory have indicated that the postribosomal supernatant of wheat germ contains a ribosome dissociation factor activity which can be detected by its ability to prevent the reassociation of ribosomal subunits at elevated a Abbreviations used: GTP, guanosine triphosphate; Hepes, 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid; BD-cellulose, benzoylated diethyl aminoethyl cellulose. 0003-2697/79/040238-06502.00/0 Copyright © 1979by AcademicPress, Inc. All rights of reproduction in any form reserved.

238

concentrations of magnesium (3-5 mM). This activity can be detected by sucrose gradient sedimentation analysis. However, the determination of ribosome dissociationfactor activity by gradient centrifugation is time consuming and can only be carried out on a limited number of samples. A simple rapid assay procedure for the ribosome dissociation factor is essential in order to follow its purification and to examine the characteristics of the reaction. Recently, Thompson et al. (10) have developed a method which measures the extent to which a rat liver factor dissociates 80 S ribosomes, or prevents the reassociation of subunits, by determining the amount of free 60 S subunits resulting or remaining in the reaction. This method depends on the ability of 60 S subunits in alcoholic solutions to catalyze the peptidyltransferase reaction between N-blocked aminoacyl-tRNA and puromycin. The N-acylpuromycin produced is then determined after extraction into ethyl acetate. In this communication we report a rapid and quantitative method for determining

RIBOSOME DISSOCIATIONFACTORASSAY

239

at 0°C in the presence of 20 mM N-ethylmaleimide. The N-ethylmaleimide was inactivated by the addition of 50 mM /3mercaptoethanol. This treatment greatly reduces the ability of elF-2 to form a stable ternary complex with Met-tRNAfmet and MATERIALS AND METHODS GTP, but does not inhibit the factor's ability to bind the initiator tRNA to the 40 S Materials. Wheat germ was kindly supribosomal subunit (12). Thus N-ethylplied by J. M. de Rosier of the International maleimide treatment reduces the backMultifoods Corporation. It was stored at ground radioactivity retained on the filter -20°C under a vacuum in the presence of a due to ternary complex formation. desiccant. Hepes and N-ethylmaleimide Preparation of tRNA. Yeast tRNAfmet were purchased from Sigma; AUG came was partially purified and separated from from Miles Laboratories. BD-Cellulose and tRNAm m~t by chromatography on BD-celluyeast tRNA were from Schwarz/Mann. lose according to the procedure of Walker [35S]Methionine was purchased from New and RajBhandary (13). Aminoacyl-tRNA England Nuclear and was diluted with unlabeled methionine to a specific activity synthetases free of tRNA were prepared from Escherichia coli as described by of about 10 Ci/mmol. Preparation of wheat germ dissociation Muench and Berg (14). [35S]Met-tRNAf met factor, ribosomes, and ribosomal subunits. (10,000-16,000 cpm/pmol) was prepared as A wheat germ ribosome dissociation-factor described previously (13) and isolated by the activity is found in the 0-40% ammonium procedure of Ravel and Shorey (15) except sulfate fraction of the postribosomal super- that the second ethanol precipitation was natant (11). This activity was partially omitted and the aminoacyl-tRNA was appurified by chromatography on DEAE- plied to Sephadex G-25 without prior cellulose and phosphocellulose as described storage. Assay for ribosomal dissociation-factor previously (11). Wheat germ high-salt activity. Assay 1 : Sucrose gradient ultracenwashed ribosomes and ribosomal subunits trifugation (11): In this analysis 0.6 Az~0unit were prepared as described previously (12). of high-salt washed ribosomes from wheat Preparation of elF-2. Wheat germ initiagerm were incubated for 5 min at 37°C in a tion factor elF-2 was purified from the reaction mixture (0.1 ml) containing 18 mM 40-60% ammonium sulfate fraction of the postribosomal supernatant by chromatog- H e p e s - K O H , pH 7.6, 5 mM /3-mercaptoraphy on DEAE-cellulose and phospho- ethanol, 9% glycerol, 0.08 mM EDTA, 70 mM cellulose (12). This elF-2 preparation is KC1, 1 mM MgCI2, and enzyme as indicated. estimated to be greater than 50% pure and The Mgz+ concentration was then raised to to be free of the other known wheat germ 5 mM by the addition of 10/zl of 45 mM MgCI~ initiation factors (12). It was tested for the and incubation was continued for an addiability to form a ternary complex with tional 5 min at 37°C. At the end of this inMet-tRNAfm~tand GTP and for its ability to cubation, the reaction mixtures were chilled bind Met-tRNAfm~t to 40 S ribosomal sub- in an ice-water bath and layered onto 5.2-ml units. The eIF-2 was treated with N-ethyl- 5-30% (w/v) linear sucrose gradients conmaleimide prior to use in the 40 S binding taining 20 mM Tris-HC1, pH 7.6, 80 mM assays or in the two-stage dissociation- KC1, 3 mM /3-mercaptoethanol, and 5 mM factor assay described below. A phospho- MgCI2. After centrifugation for 90 min at cellulose preparation containing elF-2 (54 48,000 rpm in a Beckman SW 50.1 rotor, /zg in 0.10 ml) was incubated for 10 min the uv absorbance profiles of the gradients ribosome dissociation-factor activity which takes advantage of the observation that initiation factor elF-2 catalyzes the binding of Met-tRNA~ ~t to 40 S subunits but not to 80 S ribosomes.

240

RUSSELL AND SPREMULLI

were analyzed as described previously to determine the percentage dissociation (11). Assay 2: Stage 1 : Reaction mixtures (0.1 ml) contained 0.6A~60 unit of wheat germ highsalt washed ribosomes, 18 mM H e p e s K O H , p H 7.6, 1 mM Mg(OAc)2, 70 mM KCI, 5 mM fl-mercaptoethanol, 0.08 mM E D T A , 9% glycerol, and a preparation containing the partially purified dissociation factor as indicated. Unless otherwise specified incubation was at 37°C for 5 min. Stage 2: The Mg 2+ concentration was raised to 4 mM and the amount of 40 S subunits remaining dissociated was measured by determining the amount of [3~S]Met-tRNAfmet that could be bound to the free 40 S subunits by elF-2. The second stage (0.15 ml) contained the following final concentrations: 30 mM H e p e s - K O H , p H 7.6, 3 mM /3-mercaptoethanol, 1.6 mM dithiothreitol, 0.07 mM spermine, 4 mM Mg(OAc)2, 80 mM KC1, 0.08 A260 unit of A U G , 0.07 mM GTP, 10 p m o l [35S]Met-tRNAfm~t (10,000-16,000 cpm/pmol), and 5/zg of a phosphocellulose preparation of elF-2. After incubation for 5 min at 26°C, the reaction was terminated by the addition of 2 ml of ice-cold buffer containing 20 mM T r i s - H C 1 , p H 7.6, 100 mM KC1, 5 m u /3-mercaptoethanol, and 4 mM Mg(OAc)2 and passed through a nitrocellulose filter (Millipore T y p e HA, 0.45 /xm). The filter was washed twice with 2-ml aliquots of the same buffer, dried for 5 min at 110°C, and then counted in a scintillation counter in toluene containing 5 g of 2,5-diphenyloxazole per liter. RESULTS

AND DISCUSSION

Initiation factor elF-2 is responsible for the binding of Met-tRNAf m~t to 40 S ribosomal subunits. The complex formed is retained by a Millipore filter and thus can be readily quantitated. As indicated in Fig. 1, under our assay conditions, the binding of [3~S]Met-tRNAfmet is proportional to the n u m b e r of 40 S ribosomal subunits present o v e r a wide concentration range. There is no binding to 80 S ribosomes. Wheat germ 80 S

-5

E

z

I

:3 0 m

0.6

~

o.4

z

I

I

I

"T

I

014

0.6

0.8

3~ 0.2 I

0.2

~

RIBOSOMES, A26 0 F1G. 1. The elF-2 directed binding of [asS]Met-tRNAf to 40 S subunits and 80 S ribosomes. Binding was carried out as described under Materials and Methods except that the first incubation contained 40 S subunits (©) or 80 S ribosomes (0) in the amounts indicated. The numbers have been corrected for the amount of [3~S]Met-tRNAfbound in the absence of elF-2 (0.07 pM for reactions containing 40 S subunits and 0.04 pM for reactions containing 80 S ribosomes). ribosomes can be dissociated into 40 S and 60 S subunits by incubation at 1 mM Mg 2+. When a second incubation is carried out at 4 mM Mg 2+, the subunits spontaneously reassociate to form 80 S ribosomes (11). I f the incubation at 1 mM Mg 2+ contains a partially purified ribosome dissociation factor, a certain portion of the subunits fail to reassociate when the Mg 2+ concentration is raised to 4 mM. The amount of 40 S subunits remaining dissociated due to the presence of the dissociation factor can be m e a s u r e d in the Millipore filter assay by the ability to bind Met-tRNAf met in the p r e s e n c e of elF-2. The n u m b e r of 40 S subunits remaining in the second incubation is proportional to the a m o u n t of extract containing dissociationfactor activity present in the first incubation. H e n c e , as indicated in Fig. 2, the amount of [3~S]Met-tRNAfmet bound in the second incubation is dependent on the amount of dissociation factor present in the first incubation. Analysis of the two-stage reaction mixture on sucrose gradients has confirmed that the radioactivity retained on the Millipore filter is indeed bound to the 40 S ribosomal subunit. The two-stage Millipore

241

RIBOSOME DISSOCIATION FACTOR ASSAY TABLE 1

0 E

EFFECT OF THE DISSOCIATION-FACTOR PREPARATION ON THE BINDING OF M e t - t R N A f met TO RIBOSOMES

W z 0.8

C o m p o n e n t s added u.l ~

First incubation ~

0.6

Second incubation b

[35S]Met-tRNA b o u n d (pmol)

Z

Oc 0.4

40 80 80 40 80 40 80

7 45 0.2 (,/3 I

I

I

I

I O0 200 PROTEIN,Jug FIG. 2. Effect of increasing a m o u n t s of partially purified dissociation factor on the a m o u n t of [35S]Mett R N A f b o u n d in the second stage. Incubation mixtures were prepared as described u n d e r Materials and Methods.

binding assay is more rapid than ultracentrifugation analysis. It is also quite sensitive. Figure 3 illustrates the dissociation of wheat germ ribosomes by the partially purified factor as analyzed by sucrose-gradient ultracentrifugation. This procedure requires three to four times more dissociation factor for reliable quantitation of ribosome dissociation with standard uv-scanning equipment. Table 1 summarizes the effects of the preparation containing the dissociation facIA40S

+ + + + +

3

0.98 0.06 0.45 0.07 0.04 0.84 0.02

tor in the binding of [zsS]Met-tRNAf met to 40 S and 80 S ribosomes. No binding is obtained at the end of the second incubation if 80 S ribosomes alone are used in the first incubation. However, if the dissociationfactor preparation is present in the first incubation along with the 80 S ribosomes, substantial binding of [a5S]Met-tRNAf met is obtained at the end of the second incubation.

2 VOLUME, ml 4

+eIF-2 +eIF-2 D F c +eIF-2 DF -DF -D F +eIF-2 D F +eIF-2 - A U G

a T h e first incubation was prepared as described under Materials and M e t h o d s and contained where indicated 51/~g of the partially purified dissociation factor (11), 40 S subunits (0.6 A2~0 unit) or 80 S ribosomes (0.6 A26o unit). b W h e r e indicated, 5/~g o f a phosphocellulose preparation ofeIF-2 was added. Unless otherwise specified, reaction mixtures contained 0.08 Azr0 unit o f A U G . c Dissociation factor.

BOS

60S

o.41I l

2

S S S S S S S

60S

3

4

FIG. 3. Sucrose-gradient ultracentrifugation analysis o f ribosome dissociation by the partially purified wheat germ factor. Incubations were prepared and analyzed as described u n d e r Materials and M e t h o d s and contained (A) 102 or (B) 204/~g o f the partially purified factor. T h e pattern obtained by r i b o s o m e s alone (0.4 Azr0 unit) is indicated by the dashed line in panel (A).

242

RUSSELL AND SPREMULLI '6 E A.

I

r

i

B.

i

I

C.

i

i

I

I

I

Z

0.6 m

z

0.4

n,"

i

~

T .m

0.2 .

I

I

t

3 Mg+2,mM

I

5

I

I

I

0.1 0.2 SPERMINE, mM

.06

.10 .14 KCI, M

FIG. 4. Effect of ionic conditions in the first incubation on the dissociation-factor assay. (A) Effect of Mg2+ concentration. Incubation mixtures were prepared as described under Materials and Methods except that the first incubation contained the indicated concentration of Mg2+. The second incubation contained 4 mM Mg2+ in all cases. A control containing no dissociation factor in the first incubation was performed at each concentration of Mg2+. These values (0.04 to 0.06 pmol) have been subtracted from each number. (B) The effect of spermine. The first incubation was carried out at 1 mM Mg2+ with the indicated amounts of spermine. The second incubation contained 0.15 m i spermine in all cases. A control containing no dissociation-factor preparation was performed at each concentration of spermine and this number (0.04 to 0.09 pmol) has been subtracted from each value. (C) Effect of KC1 concentration. The first incubation was carried out at 1 mM Mg~+ and the indicated KC1 concentration. The second incubation contained 80 to 100 mM KC1. The amount of [35S]Met-tRNArmet bound (0.06 to 0.1 pmol) at each KCt concentration when no dissociation factor was present in the first incubation is subtracted from each value. Reaction mixtures contained 102/zg of the partially purified dissociation-factor preparation.

The dissociation-factor preparation by itself to the structure of the ribosomes at very low cannot catalyze the binding of Met-tRNAfmet magnesium ion concentrations. Magnesium to either 40 S or 80 S ribosomes. This bind- concentrations greater than 2 mM drastically ing is strictly dependent on the presence of reduce the amount of Met-tRNAemet bound elF-2. The dissociation factor does not stimulate the elF-2 mediated binding of the -5 I I I I E initiator tRNA to 40 S subunits. The binding cS is strictly dependent on the trinucleotide z 0.6 AUG to stabilize the interaction between the E) O initiator tRNA and the 40 S subunit. This is £D especially important for elF-2 that has been ~0.4 z treated with N-ethylmaleimide to reduce the amount of ternary complex formed. Poly(A, 0,2 U,G) at a level of 6 to 10/zg can replace AUG in the second incubation if it is added after I I I I the Mg 2+ has been raised to 4 mM to permit I0 20 30 40 reassociation of the free 40 S and 60 S TEMPERATURE,°C subunits. FIG. 5. Effect of temperature in the first incubation The conditions of the first incubation proon the dissociation-factor assay. Reaction mixtures foundly effect the amount of [~sS]Met- were prepared as described under Materials and tRNAf met bound in the second stage. The Methods and contained 102 /zg of the partially puriMg 2+ concentration is especially important. fied dissociation-factor preparation. The first incubaAs indicated in Fig. 4A, the magnesium opti- tion was carried out at the indicated temperatures. The mum for the first incubation is 1 mM. Con- second incubation was at 26°C in all cases. A control containing no dissociation factor in the first incubation centrations of magnesium of less than 1 mM was carried out at each temperature and these numbers in the first incubation result in reduced bind- (0.05 to 0.09 pmol) have been subtracted from each ing in the second. This may reflect damage value. ID

i re)

t

RIBOSOME DISSOCIATION FACTOR ASSAY

in the second incubation. This may be due to the failure of the 80 S ribosome to dissociate in the first incubation at these higher Mg2+ concentrations and suggests that the wheat germ ribosome dissociation factor, like E. coli IF-3, actually acts to prevent the reassociation of ribosomal subunits (16). Polyamines such as spermine and spermidine generally reduce the dissociation of ribosomes but they have also been shown at low concentrations to enhance the binding of IF-3 to the 30 S subunit (17). For this reason, the effect of spermine in the first incubation was tested. As is indicated in Fig. 4B, even low concentrations of spermine in the first incubation greatly reduce the amount of 40 S subunits available in the second incubation. Spermine is routinely included in the second incubation since it enhances the eIF-2 mediated binding of Met-tRNAfmet to 40 S subunits. The KC1 optima for the incubation is broad (Fig. 4C) with a maximum of around 60-80 mM. Only at very high concentrations of KC1 (160 mM), in the first incubation, is there a great reduction in the degree of ribosome dissociation. The dissociation-factor activity is rapid at elevated temperatures. Maximal binding of [~sS]Met-tRNAfmet in the second incubation can be attained after 5 min at 37°C in the first incubation. The first incubation is very sensitive to temperature (Fig. 5). The dissociation factor has little activity if incubated with ribosomes at 0°C but is quite active if incubation is carried out at elevated temperatures, especially 37 or 42°C. This is in agreement with results reported for other eukaryotic ribosome dissociation factors (1,2) and for E. coli IF-3 (16). The two-stage assay outlined above allows the rapid and sensitive detection of eukaryotic ribosome dissociation-factor activity. This procedure is as rapid as that developed by Thompson et al. (10) and avoids the requirement for ethylacetate extraction of each sample. The reaction measured here is physiological and is dependent on the ability of eIF-2 to bind the initiator tRNA to 40 S subunits but not to 80 S ribo-

243

somes. This assay permits the determination of dissociation-factor activity in large numbers of samples and reduces the time required for analysis from 6 to 7 h to less than 1 h. It has already greatly facilitated the further purification of the wheat germ ribosome dissociation factor in this laboratory. ACKNOWLEDGMENTS We thank Dr. Joanne M. Ravel for helpful discussions. This work was supported in part by funds from Research Corporation and the National Institutes of Health Grant GM 24963.

REFERENCES 1. Decroly, M., and Goldfinger, M. (1975)Biochim. Biophys. Acta 390, 82-93. 2. P6tre, J. (1970) Eur. J. Biochem. 14, 399-405. 3. Thompson, H., Sadnik, I., Scheinbuks, J., and Moldave, K. (1977)Biochemistry 16, 2221-2230. 4. Lubsen, W., and Davis, B. (1974) Biochim. Biophys. Acta 335, 196-200. 5. Merrick, W., Lubsen, W., and Anderson, W. (1973) Proc. Nat. Acad. Sci. USA 70, 2220-2223. 6. Mizuno, S., and Rabinowitz, M. (1973)Proc. Nat. Aead. Sci. USA 70, 787-791. 7. Chen, Y., Woodley, C., Chatterjee, B., Majumdar, A., Milbrandt, J., and Gupta, N. (1974) Fed. Proe. 33, 1262. 8. Nakaya, K., Ranu, R., and Wool, I. (1973) Biochem. Biophys. Res. Commun. 54, 246-255. 9. Williams, G., and Maser, J. (1973) Biochem. Biophys. Res. Commun. 53, 52-58. 10. Thompson, H., Sadnik, I., and Moldave, K. (1976) Biochem. Biophys. Res. Commun. 73, 532-538. 11. Russell, D., and Spremulli, L. (1978) J. Biol. Chem., 253, 6647-6649. 12. Spremulli, L., Walthall, B., Lax, S., and Ravel, J. (1977) Arch. Biochem. Biophys. 178, 565-575. 13. Walker, R., and RajBhandary, U. (1972) J. Biol. Chem. 247, 4879-4892. 14. Muench, K., and Berg, P. (1966) in Procedures in Nucleic Acid Research (Cantoni, G., and Davies, D., eds.), p. 375, Harper & Row, New York. 15. Ravel, J., and Shorey, R. (1971) in Methods in Enzymology (Grossman, L., and Moladave, K., eds.), Vol. 20, Part C, pp. 306-316, Academic Press, New York. 16. Grunberg-Manago, M., and Gros, F, (1977) in Progress in Nucleic Acid Research and Molecular Biology (W. Cohn, ed.), Vol. 20, pp. 209-284, Academic Press, New York. 17. Pon, C., and Gualerzi, C. (1976) Biochemistry 15, 804-811.