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[43] Dissociation and reassociation of ribosomes from eukaryotic cells
[43]
DISSOCIATION OF EUKARYOTIC CELL RIBOSOMES
417
center for peptidyl transfer but also quite strongly on the presence and correct conformation of the SPs0-~ proteins that are not essential for peptide bond formation.
[43] Dissociation and Reassociation of Ribosomes from Eukaryotic Cells
By TERENCE E.
MARTIN, IRA G. WOOL, and JAMES J. CASTLES
Bacterial ribosomes readily dissociate into subunits if the concentration of magnesium is lowered, 1 but the ribosomes of many eukaryotic cells, including those from mammals, do not. When mammalian ribosomes are suspended in low concentrations of magnesium (0.01-0.1 mM) the proportion of polysomes is greatly reduced and a complex of ribosomal particles of 50-70 S is f o r m e d - c o m p l e t e dissociation requires the addition of a chelating agent. 2"3 However, subunits prepared in that way do not recombine to form 80 S monomers, nor are they active in protein synthesis even in the presence of added template RNA. 2'4,~ We have found that high concentrations of potassium cause the dissociation of cytoplasmic ribosomes of animals, plants, fungi, and protozoa into subunits capable of recombining to form monomers which, in the cases we have tested, synthesize protein in the presence of template RNA. 5-s Media
As in most experiments with ribosomes, a certain flexibility in the constitution of media is permissible, but we have generally used the following: Medium A (for intact ribosomes): 50 mM tris (hydroxymethyl) aminomethane (Tris).HC1, pH 7.8; 12.5 mM MgCI2; 80 mM KC1 Medium B (for dissociation of ribosomes): 50 mM Tris.HCl, pH 7.8; 12.5 mM MgCI2; 880 mM KC1; 20 mM 2-mercaptoethanol. (It has been found that effective dissociation of ribosomes is best ~A. Tissi6res, D. Schlessinger, and F. Gros, Proc. Nat. Acad. Sci. U. S. 46, 1450 (1960). 2H. Lamfrom and E. Glowacki,J. Mol. Biol. 5, 97 (1962). 3y. Tashiro and P. Siekevitz,J. Mol. Biol. 11, 149 (1965). 4y. Tashiro and T. Morimoto, Biochim. Biophys. Acta 123,523 (1966). ST. E. Martin, F. S. Rolleston, R. B. Low, and I. G. Wool,J. Mol. Biol. 43, 135 (1969). 6T. E. Martin and I. G. Wool, Proc. Nat. Acad. Sci. U. S. 60, 569 (1968). 7T. E. Martin and I. G. Wool,J. Mol. Biol. 43, 151 (1969). ST. E. Martin, unpublished results (1969).
418
RIBOSOME STRUCTURE AND FUNCTION
[43]
achieved with concentrations of KC1 of 0.5-1.0 M; at higher concentrations, 1.5-2.0 M, some degradation of the subunits occurs.) It should be noted that the preparation of subunits capable of reassociation requires the presence of a sulfhydryl-protecting agent during dissociation and isolation in medium B, and during dialysis against medium A (see below). Generally, 20 mM 2-mercaptoethanol is used. To avoid degradation of ribosomal RNA the sucrose used in preparation of subunits should be either commercial ribonuclease-free grade, or should be pretreated by heating of the stock sucrose solution (in water) with Norit A. a
Preparation of Ribosomes Generally we have employed standard procedures for the isolation of ribosomes. 5'7 It is economical to choose centrifugation conditions that yield the maximum number of clean ribosomal particles; methods which select the larger polyribosomes will reduce the number of ribosomes which dissociate in 0.5-1.0 M KCI. Prior preincubation to remove m R N A and nascent protein is required in order to obtain subunits from polysomes (see below). Most ribosome preparations can be stored as frozen pellets at --20°; however, ribosomes from higher plants are an exception, they appear to be inactivated by freezing and thawing. Just prior to the experiments, ribosome pellets are suspended in medium A by gentle homogenization and the suspension clarified by centrifugation for 10 min at 10,000 g. The purity of the ribosomal suspensions is assessed by determination of the absorbancy at 235, 260, and 280 m~. 1° Only those ribosomal preparations having absorbancy ratios, 260:235 and 260:280, greater than 1.45 and 1.85, respectively, are used for preparations of subunits; lower ratios generally indicate the presence of impurities which interfere with the resolution of the subunits on sucrose density gradients.
Dissociation of Ribosomes by High Potassium Concentrations Preparation of Ribosomal Subunits. After suspension of ribosomes in medium A and clarification, the KCI concentration is adjusted to 0.5-1.0 M - a convenient concentration is 880 m M - a n d 2-mercaptoethanol is added to 20 mM (medium B). When a Spinco SW 27 rotor is used, the suspension (1-2 ml containing 40-80 OD2~0 units) is layered on to a 35 ml 10-30% linear sucrose density gradient containing medium B. Centrifugation is at 27,000 rpm, and the time required 9W. S. Stirewalt, I. G. Wool, and P. Cavicchi, Proc. Nat. Acad. Sci. U. S. 57, 1885 (1967). I°M. L. Petermann, " T h e Physical and Chemical Properties of Ribosomes," American Elsevier, New York, 1964.
[43]
419
DISSOCIATION OF EUKARYOTIC CELL RIBOSOMES
d e p e n d s on the t e m p e r a t u r e o f the run, and this is a function o f the source o f the ribosomes (see below): at 28 ° centrifugation is for 3.5 hours, at 4 ° for 8 hours. If large quantities o f ribosomal subunits are required, a zonal r o t o r (Spinco Ti 15) can be used for the preparation. T h e ribosome suspension (5,000-15,000 ODzn0 units in 40 ml o f 5% sucrose in m e d i u m B) is layered on to 1200 ml o f a 10-30% linear sucrose gradient in m e d i u m B and centrifuged at 20,000 r p m for 16.5 hours at 28 °. A typical absorbancy pattern obtained f r o m the centrifugation o f rat liver ribosomes in a zonal r o t o r is shown in Fig. 1. Effect of Temperature. T h e distribution o f ribosomal particles centrif u g e d on sucrose density gradients containing m e d i u m B may be affected by the t e m p e r a t u r e at which the dissociation and centrifugation is p e r f o r m e d . While rabbit skeletal muscle ribosomes dissociate into 40 S and 60 S subunits in m e d i u m B at 4 °, ribosomes f r o m rat muscle give rise to large a m o u n t s o f 90 S and 105 S particles at this t e m p e r a t u r e (Fig. 2). At 28 °, however, rat muscle ribosomes dissociate in m e d i u m B to 40 S and 60 S subunits. We believe that the 90 S and 105 S particles f o r m e d by some ribosomes at low t e m p e r a t u r e are the result o f subunit
60S de
0.7 0.5 o 0.4 0.:3 0.2 0.1
!iS
o
30
25
2o
15 e,
S '
I0
,
I
20
30
,
40
'
50
,
I
,
60
70
80
i 90
10
Fraction FIG. 1. P r e p a r a t i o n o f rat liver ribosomal s u b u n i t s in a zonal rotor. A 1200-ml linear 1 0 - 3 0 % sucrose g r a d i e n t in m e d i u m B was p u m p e d into the r o t o r (Spinco Ti 15) with a Spinco Model 141 G r a d i e n t P u m p . T h e r o t o r was t h e n filled by a d d i n g 466 ml o f 3 0 % sucrose in m e d i u m B. A p p r o x i m a t e l y 12,000 OD260 units o f r i b o s o m e s ( p r e i n c u b a t e d with p u r o m y c i n ) in 40 ml o f 5 % sucrose in m e d i u m B were layered o n t o the g r a d i e n t a n d t o p p e d with an overlay o f 80 ml o f m e d i u m B. T h e conditions o f c e n t r i f u g a t i o n are in t h e text; 20 ml fractions were collected; t h e analysis o f fractions a - f is in Fig. 4.
420
RIBOSOME STRUCTURE AND FUNCTION
Rat 4 .0
[43]
Rol 28 °
04
60S
05
0.2
60 S
I
C3 0
\9os
o lt /
Rabbi14 °
I
60S'
Rabbit 28 °
6os
().5 40S
OI /x2 FIG. 2. Dissociation of rat and rabbit skeletal muscle ribosomes at 4° and 28 °. Ribosomes (30 ~g ribosomal RNA) were analyzed on 15-30% sucrose gradients containing medium B without 2-mercaptoethanol. Centrifugation was at 4° in a SW 39 rotor at 39,000 rpm for 120 minutes, or at 28° in a SW 65 rotor at 60,000 rpm for 30 minutes. a g g r e g a t i o n ; in p a r t i c u l a r , t h e 90 S p a r t i c l e is m o s t p r o b a b l y a d i m e r o f t h e 6 0 S s u b u n i t ? ( R a t l i v e r 60 S s u b u n i t s h a v e a g r e a t e r t e n d e n c y to d i m e r i z e t h a n d o 60 S s u b u n i t s f r o m m u s c l e ; m o r e o v e r , t h e f r a c t i o n o f 60 S s u b u n i t s t h a t d i m e r i z e is c o n d i t i o n e d b y t h e m a g n e s i u m c o n c e n t r a tion and the concentration of the subunits themselves. Preincubation o f r a t r i b o s o m e s w i t h p u r o m y c i n to r e m o v e n a s c e n t p r o t e i n a p p e a r s to i n c r e a s e t h e t e n d e n c y o f b o t h m o n o m e r s a n d s u b u n i t s to f o r m d i m e r s . 8) T h e p r o p e r t y o f s u b u n i t a g g r e g a t i o n at low t e m p e r a t u r e is a p p a r e n t l y species dependent, and thus the best temperature for dissociation must be determined for the ribosomes from each source. While rabbit
[43]
DISSOCIATION
OF EUKARYOTIC
CELL RIBOSOMES
421
and rat ribosomal subunits are not greatly affected by isolation at 28 °, the subunits of some higher plants and of Tetrahymena pyriformis are inactivated at this temperature; therefore the lower temperature is to be preferred whenever practicable.
Purity of Ribosomal Subunits. The nature and purity of ribosomal components isolated from the sucrose gradients can be assessed by analysis of their constituent RNA. Sodium dodecyl sulfate is used to dissociate protein from ribosomal RNA. The gradient fractions are first dialyzed overnight against 50 mM Tris (pH 7.8) in 6% sucrose; sodium dodecyl sulfate (5% w/v in water) is then added to a concentration of 0.1%. After incubation at 37 ° for 3 minutes, the mixture is cooled in ice and a 0.2 ml sample is analyzed on a 15-30% sucrose gradient containing 50 mM Tris (pH 7.8); centrifugation is at 60,000 rpm for 3 hours in a Spinco SW 65 rotor at 4 °. [Analysis is facilitated by use of an Instrument Specialties Co., Inc. (ISCO) Model D, density gradient fractionator, and the model UA-2 UV analyzer.] The results (Fig. 3) of such an analysis of subunits of rat muscle ribosomes show that the 40 S fraction contains 18 S RNA; the 60 S fraction has predominantly 28 S RNA, but also a small amount of 18 S RNA, indicating some contamination by small subunits, and some RNA having sedimentation coefficients between 18 and 28 (the latter appears to arise from breakdown of 28 S RNA). The 75 S fraction obtained from ribosomes centrifuged in medium B contains 18 S and 28 S RNA and thus apparently consists of undissociated ribosome monomers. The nature of the ribosomal components isolated from preparative gradients can also be determined on analytical gradients. The fractions collected from the preparative sucrose gradients are dialyzed overnight against medium A; 0.5-1.0 OD260 units are layered on to a 15-30% sucrose gradient in medium A and centrifuged at 60,000 rpm for 40 minutes at 28 ° in a Spinco SW 65 rotor. The results of analysis of fractions collected from a preparative gradient of preincubated rat liver ribosomes centrifuged in a zonal rotor shows that the slower sedimenting peak (Fig. 1) contains 40 S subunits (Fig. 4a and b); fraction a probably is uncontaminated, whereas fraction b contains a small amount of the 60 S subunit (manifest by the presence of 80 S ribosomes; Fig. 4b). If the fractions in the 40 S peak are collected (as indicated by the bars in Fig. 1) and pooled, they will contain 10% of the OD2n0 units of ribosomes applied to the gradient and there will be less than 5% contamination with 60 S subunits. The faster sedimenting peak (Fig. 1) contains the 60 S subunit. However, the portion of the peak that sediments closest to the center of rotation (fraction c, Fig. 1)
422
RIBOSOME S T R U C T U R E AND FUNCTION (o) 4 0 S Fraction
0.4 40S
[43]
[d)40 S Fraction
RNA
18S
0.3
0.2 0.1
I(b)60 S Fraction
o.41
(e)60 S Fraction RNA "28 S
0.31ea 123
o
I
0.1
(c) 75 S Fraction 0.4
(f) 75 S Fraction RNA
0.3
/
0.2
~60S
75s
/
28S
FIG. 3. T h e s e d i m e n t a t i o n o f R N A f r o m t h e 40 S, 60 S, a n d 75 S fractions o f rat skeletal m u s c l e ribosomes. R i b o s o m e s were dissociated in m e d i u m B a n d f r a c t i o n a t e d at 28 ° o n 1 0 - 3 0 % sucrose gradients, c o n t a i n i n g t h e s a m e buffer. T h e r i b o s o m e fractions were collected a n d t h e i r purity was d e t e r m i n e d (a-c) by analysis o n 1 5 - 3 0 % s u c r o s e g r a d i e n t s c o n t a i n i n g m e d i u m B w i t h o u t 2 - m e r c a p t o e t h a n o l . T h e a b s o r p t i o n at t h e t o p o f t h e s e g r a d i e n t s is d u e to 2 - m e r c a p t o e t h a n o l r e m a i n i n g f r o m the p r e p a r a t i v e m e d i u m . C e n t r i f u g a t i o n was at 28 ° for 40 m i n u t e s at 60,000 r p m in a SW 65 rotor (a-c). T h e s e d i m e n t a t i o n o f t h e R N A (d-f) isolated f r o m t h e fractions was d e t e r m i n e d as d e s c r i b e d in t h e text.
[43]
DISSOCIATION OF EUKARYOTIC CELL RIBOSOMES
(a)
"(b)
4O
0
T (e)
60 8O
80
(c)
I
4O
-(d)
423
60
(f)
6O
90
I
F~G. 4. Analysis o f rat liver ribosome s u b u n i t fractions. T h e fractions are f r o m the g r a d i e n t s h o w n in Fig. l a n d were collected at t h e points indicated by the arrows. Analysis was as described in t h e text.
is heavily contaminated with 40 S subunits (Fig. 4c), which is manifest by the presence of a large a m o u n t of 80 S ribosomes (the result of reassociation of subunits in m e d i u m A). T h e a m o u n t of contamination is decreased when the fractions are collected f u r t h e r from the center of rotation (Fig. 4 c-f); fractions e and f contain relatively small amounts of 40 S subunits (as reflected in the small amounts of 80 S and 105 S particles) and large numbers of 60 S subunits (i. e., 60 S and 90 S particles). T h e contamination of the 60 S peak is primarily the result of sedimentation of 40 S subunits as 55 S particles in m e d i u m B (Fig. 5). We do not know if the 55 S particle is a dimer or a more compact form of the 40 S subunit. If only that portion of the 60 S peak indicated by the bars (Fig. 1) is collected, it will contain 20-35% of the OD2~0 units applied to the gradient and will be contaminated with 5-10% of 40 S subunits. We have been unable to obtain mammalian 60 S subunits completely free of 40 S particles. Ribosomes Resistant to Dissociation. When ribosomes containing nascent protein labeled with radioactive amino acids in vitro are dissociated by
424
RIBOSOMES S T R U C T U R E AND F U N C T I O N
[43]
55S
O
¢,,
FIG. 5. S e d i m e n t a t i o n o f 40 S liver ribosomal s u b u n i t s in m e d i u m B. 40 S s u b u n i t s (fraction a o f Figs. 1 a n d 4) were dialyzed against m e d i u m B a n d t h e n layered o n t o 1 5 - 3 0 % linear s u c r o s e g r a d i e n t in m e d i u m B. C e n t r i f u g a t i o n was at 60,000 r p m for 40 m i n u t e s at 28 ° in a SW 65 rotor.
KCI, the greater part of the radioactivity remains attached to the 75 S component (Fig. 6). Thus it appears that ribosomes active in the synthesis of protein on endogenous messenger RNA are more resistant to dissociation. This conclusion is supported by the effect of preincubation of ribosomes to remove nascent protein and messenger RNA. Untreated rat liver ribosomes contain a greater proportion of monomers resistant to dissociation than do preparations from rat skeletal muscle (compare Figs. 2 and 7a). The proportion of the resistant 75 S particles is reduced by preincubation of the ribosomes with factors required for protein synthesis (Fig. 7b). If puromycin, which releases nascent peptide from ribosomes, is included in the preincubation medium, the particles completely dissociate at high concentrations of KC1 (Fig. 7c). Since the large proportion of 75 S particles that remain after dissociation of untreated liver ribosomes (Fig. 7a) results in low yields and causes inevitable contamination of the large subunit fraction, it is convenient to preincubate liver ribosomes with puromycin before preparation of the subunits. Preincubation can be carried out for 30 minutes at 37 ° in 60 ml of medium A containing 20 mM 2-mercaptoethanol, 5 mg of puromycin, approximately 6 0 0 r a g of liver supernatant protein, and 5000 OD~e0 units of ribosomes. After preincubation the ribosomes are reisolated in the customary way. It must be pointed out, however, that preincubafion with puromycin may modify the ribosomes, e. g., the
[43]
425
DISSOCIATION OF EUKARYOTIC CELL RIBOSOMES
(a) 80 m/W KCI
3000
0.6 2000 80S
0.4
I000 0.2
'~"
.£ E
tO
60S
I
(b) 880 mM KCI
C,
3000
0.6~ 2000
0.4
0,2
%
I000
I
I
FIG. 6. T h e presence o f nascent protein in the 75 S fraction o f rat skeletal muscle ribosomes dissociated by KCI at 28 °. Ribosomes ( 1 m g ribosomal RNA/ml) were incubated at 37 ° for 30 minutes in m e d i u m containing 50 m M Tris-HC1, p H 7.8, 80 m M KCI, 12.5 m M MgCIz, 5 m M ATP, 0.5 m M GTP, 1 m M phosphoenolpyruvate, 100 p,g/ml aminoacyl-tRNA containing 59,000 c p m [3H]phenylalanine. T h e reaction mixture (2 ml) was centrifuged on a 15-30% sucrose gradient containing: (a) m e d i u m A; (b) m e d i u m B without 2-mercaptoethanol. Centrifugation was at 28 ° for 5 hours at 22,500 r p m in a SW 25.1 rotor. T h e radioactivity o f the protein contained in l-ml fractions from the gradients was d e t e r m i n e d .
tendency to dimerizes and the magnesium dependency of polyphenylalanine synthesis.
426
RIBOSOMES STRUCTURE AND FUNCTION
0.4
(a) Untreated 75 S
(b) Preincubated 60 S
[43]
(c) Stripped
(puromycin) 60 S
0.3 0
60i/
0.2 O. I
40~ ~
F1c. 7. Effect o f preincubation and puromycin on the dissociation o f rat liver ribosomes. Rat liver ribosomes were (a) p r e p a r e d in the usual m a n n e r , (b) then incubated at 37 ° for 30 minutes in the m e d i u m for the assay o f protein synthesis, or (c) incubated in the m e d i u m for the assay o f protein synthesis containing in addition 5 0 / z g / m l puromycin; the p r e i n c u b a t e d ribosomes (b,c) were collected by centrifugation. T h e ribosomes (30/~g ribosomal RNA) were analyzed on 15-30% sucrose gradients containing m e d i u m B without 2-mercaptoethanol. Centrifugation was in a SW 65 rotor at 60,000 r p m for 30 minutes at 28 ° .
Reassociation of Ribosomal Subunits The 40 S and 60 S subunit fractions collected from sucrose gradients may be treated separately or mixed to prepare reassociated ribosomes. For reassociation, subunit fractions are combined in the ratio (60 S:40 S) of approximately 2.5:1 (ODin0); the proportions in which they are formed. 5 The separate subunit fractions or the combined subunits are then dialyzed overnight at 4 ° against medium A containing 20 mM
40S Subunit
60 SSubunil
Reossociated 80 S
0.5 40 ea
60 S
0.2 0.1
FIG. 8. T h e reassociation o f subunits o f rat skeletal muscle ribosomes. Separate and c o m b i n e d ribosomal subunits were dialyzed against m e d i u m A containing 20 m M 2m e r c a p t o e t h a n o l a n d analyzed on 15-30% sucrose gradients containing m e d i u m A. Centrifugation was at 60,000 r p m in a SW ,65 r o t o r for 30 minutes at 28 °.
[43]
427
DISSOCIATION OF EUKARYOTIC CELL RIBOSOMES
2-mercaptoethanol. After dialysis, the particles are analyzed by centrifugation on sucrose gradients containing medium A. The above procedure can lead to the complete reassociation of ribosomal subunits (Fig. 8). Consistently high levels of reassociation have been observed with rat, mouse, rabbit, yeast, and Tetrahymena pyriformis ribosomal subunits: wheat and pea have generally given lower yields (approximately 50%) apparently due to the greater lability of plant ribosomal subunits, particularly of the small subunit, s If ribosome subunit preparations from different sources are used, active hybrid 80 S particles may be obtained, n'7
Activity of the Subunits and of Reassociated Ribosomes When separate ribosomal subunit fractions or reassociated ribosomes are assayed for their ability to synthesize protein in a cell-free system very low activities are observed in the absence of added template RNA (see the table). The 40 S fraction is almost totally inactive even when assayed with polyuridylic acid, whereas the 60 S fraction has approxiPROTEIN SYNTHESIS BY SUBUNITS FROM RAT MUSCLE RIBOSOMES
Protein synthesis u (cpm/10/~g ribosomal RNA) Experimental conditions
Endogenous synthesis
Polyphenylalanine synthesis ~
527 307 0 16 18 18 377
4690 4593 178 1094 5415 4115 276
734 512 10 25 67
5910 4355 210 1465 6553
A. Assay after dialysis Untreated ribosomes KCl-treated ribosomes 40 S fraction 60 S fraction 40 S + 60 S mixed before dialysis 40 S + 60 S mixed after dialysis 75 S fraction
B. Direct assay Untreated ribosomes KCl-treated ribosomes 40 S fraction 60 S fraction 40S + 60S
~'The medium for the assay of protein synthesis contained (in 1 ml): 50 mM Tris.HC1 (pH 7.8), 88 mM KCI, 15 mM MgCI~, 10 mM 2-mercaptoethanol, 5 mM ATP, 0.4 mM GTP, 1 mM phosphoenolpyruvate, 10/~g of pyruvate kinase, 1.5-2% sucrose, 2 mg of muscle supernatant protein, and 100 ~g of aminoacyl-transfer RNA containing 30,000 cpm of [aH]phenylalanine. The amount of the ribosomal particles (ribosomal RNA) was 7.5-12.5 /zg, and the amount of polyuridylic acid added was 100 ~g. Incubation was at 37 ° for 30 minutes. Each 100 cpm in protein was the result of the incorporation of 2.3 x 10-13 moles of phenylalanine. bCorrected for endogenous incorporation of phenylalanine.
428
RIBOSOME STRUCTURE AND FUNCTION
[43]
mately 20% of the activity of the combined fractions (see the table), which accords with the presence of some 40 S particles in the 60 S fraction (Figs. 3 and 4). In the presence of polyuridylic acid the combined 60 S and 40 S fractions possess activity equal to, or exceeding that of the control ribosomes. The results are more consistent if the subunits are combined before dialysis, although there is activity if the subunits are combined after separate dialysis (part A of table). Activity is also restored if the subunit fractions are mixed, then assayed (without first being dialyzed) in medium whose ionic composition is adjusted to compensate for the excess of KC1 contained in the fractions (part B of table, direct assay). Rapid and specific reassociation of the subunits must occur in the reaction mixture. The direct assay, however, gives somewhat variable results and suffers the additional limitation that the products of reassociation cannot easily be analyzed on sucrose gradients. For these reasons ribosomal subunit fractions are generally dialyzed against medium A containing 20 mM 2-mercaptoethanol before assay of protein synthesis. Unlike the reassociated ribosomes which are inactive in the absence of added template RNA, the 75 S particles have almost the same endogenous activity as the control ribosomes; moreover, they are relatively unresponsive to polyuridylic acid (part A of table). These observations provide additional support for the view that the 75 S particles derive from ribosomes active in endogenous protein synthesis. The specific endogenous protein synthetic activity (cpm incorporated into protein per microgram of ribosomal RNA) of the 75 S fraction is not considerably greater than the original ribosomal preparation as would be expected if inactive particles have been removed by dissociation; thus, the experimental procedure must have caused some inactivation of the 75 S particles, although how much is not certain since the fraction does contain 60 S particles which do not have endogenous activity and would therefore lower the specific activity. The results suggest, however, that the dissociation procedure allows the separation of the ribosomes in the population that were active (the 75 S fraction) from subunits derived from ribosomes not carrying out endogenous protein synthesis, and this operation is potentially useful in the analysis of ribosome function. Remarks
The major difficulty associated with the procedure for the dissociation of ribosomes from eukaryotic cells, particularly those of mammals, is the contamination of the large subunit fraction by the small subunits.
[44]
ACTIVE SUBUNITS FROM LIVER RIBOSOMES
429
T h e contamination most likely arises f r o m the presence o f dimers or a m o r e compact f o r m o f the small subunit which cosediment with the large subunit in sucrose gradients. In addition, the 60 S subunit may be c o n t a m i n a t e d by 80 S ribosomes that are resistant to dissociation because they contain nascent peptide and messenger RNA; the latter source o f contamination can be minimized by p r e t r e a t i n g the ribosome with puromycin, although it must be realized that preincubation may slightly alter ribosome structure and f u n c t i o n / Despite the difficulty, the m e t h o d has already p r o v e d useful in locating a defect in the muscle ribosomes o f diabetic animals, 6 in determining that m R N A 11 and mRNA-directed tRNA binding 12is to the small subunit o f mammalian ribosomes, and in the investigation o f the interaction o f ribosomal subunits o f different species/ 11F.S. Rolleston, T. E. Martin, and I. G. Wool,Biochem.J. 117,899 (1970). ~.1.J. Castles and I. G. Wool,Biochemistry 9, 1909 (1970).
[44] The Dissociation of Rat Liver Ribosomes to Active Subunits by Urea By MARY L. PETERMANN W h e n rat liver ribosomes are dissociated by r e m o v i n g all their magnesium the 5 S RNA is detached and the subunits are inactive in an amino acid-incorporating system. 1 Active subunits can, however, be obtained by merely r e d u c i n g the ribosomal magnesium, then dissociating by t r e a t m e n t with u r e a / W i t h this p r o c e d u r e , precautions against d a m a g e by RNase are o f great importance. One must work rapidly, keep the material cold, and use an RNase inhibitor, such as bentonite. Bentonite. Bentonite (Fisher, USP) is equilibrated with 0.5 m M MgCI2, 1 m M potassium phosphate, p H 6.8, at 5 °, by s u s p e n d i n g 90 g in buffer in a blender, diluting the suspension to 2 liters, and stirring overnight. T h e suspension is c e n t r i f u g e d at 8000 g for 10 minutes. T h e pellets are discarded, and the s u p e r n a t a n t is used for the p r e p a r a t i o n o f "coarse" and "fine" fractions? T h e coarse bentonite is obtained by centrifuging the bentonite at 20,000 g for 15 minutes. T h e pellets are s u s p e n d e d in buffer with a pestle homogenizer, and the suspension is diluted to 1 liter, 1M. L. Petermann and A. Pavlovec,Biopolymers 7, 73 (1969). 2M. L. Petermann, A. Pavlovec,and I. B. Weinstein, Fed. Proc. Fed. Amer. Soc. Exp. Biol. 28, 725 (1969). 3M. L. Petermann and A. Pavlovec,J. Biol. Chem. 238, 3717 (1963).
DISSOCIATION OF EUKARYOTIC CELL RIBOSOMES
417
center for peptidyl transfer but also quite strongly on the presence and correct conformation of the SPs0-~ proteins that are not essential for peptide bond formation.
[43] Dissociation and Reassociation of Ribosomes from Eukaryotic Cells
By TERENCE E.
MARTIN, IRA G. WOOL, and JAMES J. CASTLES
Bacterial ribosomes readily dissociate into subunits if the concentration of magnesium is lowered, 1 but the ribosomes of many eukaryotic cells, including those from mammals, do not. When mammalian ribosomes are suspended in low concentrations of magnesium (0.01-0.1 mM) the proportion of polysomes is greatly reduced and a complex of ribosomal particles of 50-70 S is f o r m e d - c o m p l e t e dissociation requires the addition of a chelating agent. 2"3 However, subunits prepared in that way do not recombine to form 80 S monomers, nor are they active in protein synthesis even in the presence of added template RNA. 2'4,~ We have found that high concentrations of potassium cause the dissociation of cytoplasmic ribosomes of animals, plants, fungi, and protozoa into subunits capable of recombining to form monomers which, in the cases we have tested, synthesize protein in the presence of template RNA. 5-s Media
As in most experiments with ribosomes, a certain flexibility in the constitution of media is permissible, but we have generally used the following: Medium A (for intact ribosomes): 50 mM tris (hydroxymethyl) aminomethane (Tris).HC1, pH 7.8; 12.5 mM MgCI2; 80 mM KC1 Medium B (for dissociation of ribosomes): 50 mM Tris.HCl, pH 7.8; 12.5 mM MgCI2; 880 mM KC1; 20 mM 2-mercaptoethanol. (It has been found that effective dissociation of ribosomes is best ~A. Tissi6res, D. Schlessinger, and F. Gros, Proc. Nat. Acad. Sci. U. S. 46, 1450 (1960). 2H. Lamfrom and E. Glowacki,J. Mol. Biol. 5, 97 (1962). 3y. Tashiro and P. Siekevitz,J. Mol. Biol. 11, 149 (1965). 4y. Tashiro and T. Morimoto, Biochim. Biophys. Acta 123,523 (1966). ST. E. Martin, F. S. Rolleston, R. B. Low, and I. G. Wool,J. Mol. Biol. 43, 135 (1969). 6T. E. Martin and I. G. Wool, Proc. Nat. Acad. Sci. U. S. 60, 569 (1968). 7T. E. Martin and I. G. Wool,J. Mol. Biol. 43, 151 (1969). ST. E. Martin, unpublished results (1969).
418
RIBOSOME STRUCTURE AND FUNCTION
[43]
achieved with concentrations of KC1 of 0.5-1.0 M; at higher concentrations, 1.5-2.0 M, some degradation of the subunits occurs.) It should be noted that the preparation of subunits capable of reassociation requires the presence of a sulfhydryl-protecting agent during dissociation and isolation in medium B, and during dialysis against medium A (see below). Generally, 20 mM 2-mercaptoethanol is used. To avoid degradation of ribosomal RNA the sucrose used in preparation of subunits should be either commercial ribonuclease-free grade, or should be pretreated by heating of the stock sucrose solution (in water) with Norit A. a
Preparation of Ribosomes Generally we have employed standard procedures for the isolation of ribosomes. 5'7 It is economical to choose centrifugation conditions that yield the maximum number of clean ribosomal particles; methods which select the larger polyribosomes will reduce the number of ribosomes which dissociate in 0.5-1.0 M KCI. Prior preincubation to remove m R N A and nascent protein is required in order to obtain subunits from polysomes (see below). Most ribosome preparations can be stored as frozen pellets at --20°; however, ribosomes from higher plants are an exception, they appear to be inactivated by freezing and thawing. Just prior to the experiments, ribosome pellets are suspended in medium A by gentle homogenization and the suspension clarified by centrifugation for 10 min at 10,000 g. The purity of the ribosomal suspensions is assessed by determination of the absorbancy at 235, 260, and 280 m~. 1° Only those ribosomal preparations having absorbancy ratios, 260:235 and 260:280, greater than 1.45 and 1.85, respectively, are used for preparations of subunits; lower ratios generally indicate the presence of impurities which interfere with the resolution of the subunits on sucrose density gradients.
Dissociation of Ribosomes by High Potassium Concentrations Preparation of Ribosomal Subunits. After suspension of ribosomes in medium A and clarification, the KCI concentration is adjusted to 0.5-1.0 M - a convenient concentration is 880 m M - a n d 2-mercaptoethanol is added to 20 mM (medium B). When a Spinco SW 27 rotor is used, the suspension (1-2 ml containing 40-80 OD2~0 units) is layered on to a 35 ml 10-30% linear sucrose density gradient containing medium B. Centrifugation is at 27,000 rpm, and the time required 9W. S. Stirewalt, I. G. Wool, and P. Cavicchi, Proc. Nat. Acad. Sci. U. S. 57, 1885 (1967). I°M. L. Petermann, " T h e Physical and Chemical Properties of Ribosomes," American Elsevier, New York, 1964.
[43]
419
DISSOCIATION OF EUKARYOTIC CELL RIBOSOMES
d e p e n d s on the t e m p e r a t u r e o f the run, and this is a function o f the source o f the ribosomes (see below): at 28 ° centrifugation is for 3.5 hours, at 4 ° for 8 hours. If large quantities o f ribosomal subunits are required, a zonal r o t o r (Spinco Ti 15) can be used for the preparation. T h e ribosome suspension (5,000-15,000 ODzn0 units in 40 ml o f 5% sucrose in m e d i u m B) is layered on to 1200 ml o f a 10-30% linear sucrose gradient in m e d i u m B and centrifuged at 20,000 r p m for 16.5 hours at 28 °. A typical absorbancy pattern obtained f r o m the centrifugation o f rat liver ribosomes in a zonal r o t o r is shown in Fig. 1. Effect of Temperature. T h e distribution o f ribosomal particles centrif u g e d on sucrose density gradients containing m e d i u m B may be affected by the t e m p e r a t u r e at which the dissociation and centrifugation is p e r f o r m e d . While rabbit skeletal muscle ribosomes dissociate into 40 S and 60 S subunits in m e d i u m B at 4 °, ribosomes f r o m rat muscle give rise to large a m o u n t s o f 90 S and 105 S particles at this t e m p e r a t u r e (Fig. 2). At 28 °, however, rat muscle ribosomes dissociate in m e d i u m B to 40 S and 60 S subunits. We believe that the 90 S and 105 S particles f o r m e d by some ribosomes at low t e m p e r a t u r e are the result o f subunit
60S de
0.7 0.5 o 0.4 0.:3 0.2 0.1
!iS
o
30
25
2o
15 e,
S '
I0
,
I
20
30
,
40
'
50
,
I
,
60
70
80
i 90
10
Fraction FIG. 1. P r e p a r a t i o n o f rat liver ribosomal s u b u n i t s in a zonal rotor. A 1200-ml linear 1 0 - 3 0 % sucrose g r a d i e n t in m e d i u m B was p u m p e d into the r o t o r (Spinco Ti 15) with a Spinco Model 141 G r a d i e n t P u m p . T h e r o t o r was t h e n filled by a d d i n g 466 ml o f 3 0 % sucrose in m e d i u m B. A p p r o x i m a t e l y 12,000 OD260 units o f r i b o s o m e s ( p r e i n c u b a t e d with p u r o m y c i n ) in 40 ml o f 5 % sucrose in m e d i u m B were layered o n t o the g r a d i e n t a n d t o p p e d with an overlay o f 80 ml o f m e d i u m B. T h e conditions o f c e n t r i f u g a t i o n are in t h e text; 20 ml fractions were collected; t h e analysis o f fractions a - f is in Fig. 4.
420
RIBOSOME STRUCTURE AND FUNCTION
Rat 4 .0
[43]
Rol 28 °
04
60S
05
0.2
60 S
I
C3 0
\9os
o lt /
Rabbi14 °
I
60S'
Rabbit 28 °
6os
().5 40S
OI /x2 FIG. 2. Dissociation of rat and rabbit skeletal muscle ribosomes at 4° and 28 °. Ribosomes (30 ~g ribosomal RNA) were analyzed on 15-30% sucrose gradients containing medium B without 2-mercaptoethanol. Centrifugation was at 4° in a SW 39 rotor at 39,000 rpm for 120 minutes, or at 28° in a SW 65 rotor at 60,000 rpm for 30 minutes. a g g r e g a t i o n ; in p a r t i c u l a r , t h e 90 S p a r t i c l e is m o s t p r o b a b l y a d i m e r o f t h e 6 0 S s u b u n i t ? ( R a t l i v e r 60 S s u b u n i t s h a v e a g r e a t e r t e n d e n c y to d i m e r i z e t h a n d o 60 S s u b u n i t s f r o m m u s c l e ; m o r e o v e r , t h e f r a c t i o n o f 60 S s u b u n i t s t h a t d i m e r i z e is c o n d i t i o n e d b y t h e m a g n e s i u m c o n c e n t r a tion and the concentration of the subunits themselves. Preincubation o f r a t r i b o s o m e s w i t h p u r o m y c i n to r e m o v e n a s c e n t p r o t e i n a p p e a r s to i n c r e a s e t h e t e n d e n c y o f b o t h m o n o m e r s a n d s u b u n i t s to f o r m d i m e r s . 8) T h e p r o p e r t y o f s u b u n i t a g g r e g a t i o n at low t e m p e r a t u r e is a p p a r e n t l y species dependent, and thus the best temperature for dissociation must be determined for the ribosomes from each source. While rabbit
[43]
DISSOCIATION
OF EUKARYOTIC
CELL RIBOSOMES
421
and rat ribosomal subunits are not greatly affected by isolation at 28 °, the subunits of some higher plants and of Tetrahymena pyriformis are inactivated at this temperature; therefore the lower temperature is to be preferred whenever practicable.
Purity of Ribosomal Subunits. The nature and purity of ribosomal components isolated from the sucrose gradients can be assessed by analysis of their constituent RNA. Sodium dodecyl sulfate is used to dissociate protein from ribosomal RNA. The gradient fractions are first dialyzed overnight against 50 mM Tris (pH 7.8) in 6% sucrose; sodium dodecyl sulfate (5% w/v in water) is then added to a concentration of 0.1%. After incubation at 37 ° for 3 minutes, the mixture is cooled in ice and a 0.2 ml sample is analyzed on a 15-30% sucrose gradient containing 50 mM Tris (pH 7.8); centrifugation is at 60,000 rpm for 3 hours in a Spinco SW 65 rotor at 4 °. [Analysis is facilitated by use of an Instrument Specialties Co., Inc. (ISCO) Model D, density gradient fractionator, and the model UA-2 UV analyzer.] The results (Fig. 3) of such an analysis of subunits of rat muscle ribosomes show that the 40 S fraction contains 18 S RNA; the 60 S fraction has predominantly 28 S RNA, but also a small amount of 18 S RNA, indicating some contamination by small subunits, and some RNA having sedimentation coefficients between 18 and 28 (the latter appears to arise from breakdown of 28 S RNA). The 75 S fraction obtained from ribosomes centrifuged in medium B contains 18 S and 28 S RNA and thus apparently consists of undissociated ribosome monomers. The nature of the ribosomal components isolated from preparative gradients can also be determined on analytical gradients. The fractions collected from the preparative sucrose gradients are dialyzed overnight against medium A; 0.5-1.0 OD260 units are layered on to a 15-30% sucrose gradient in medium A and centrifuged at 60,000 rpm for 40 minutes at 28 ° in a Spinco SW 65 rotor. The results of analysis of fractions collected from a preparative gradient of preincubated rat liver ribosomes centrifuged in a zonal rotor shows that the slower sedimenting peak (Fig. 1) contains 40 S subunits (Fig. 4a and b); fraction a probably is uncontaminated, whereas fraction b contains a small amount of the 60 S subunit (manifest by the presence of 80 S ribosomes; Fig. 4b). If the fractions in the 40 S peak are collected (as indicated by the bars in Fig. 1) and pooled, they will contain 10% of the OD2n0 units of ribosomes applied to the gradient and there will be less than 5% contamination with 60 S subunits. The faster sedimenting peak (Fig. 1) contains the 60 S subunit. However, the portion of the peak that sediments closest to the center of rotation (fraction c, Fig. 1)
422
RIBOSOME S T R U C T U R E AND FUNCTION (o) 4 0 S Fraction
0.4 40S
[43]
[d)40 S Fraction
RNA
18S
0.3
0.2 0.1
I(b)60 S Fraction
o.41
(e)60 S Fraction RNA "28 S
0.31ea 123
o
I
0.1
(c) 75 S Fraction 0.4
(f) 75 S Fraction RNA
0.3
/
0.2
~60S
75s
/
28S
FIG. 3. T h e s e d i m e n t a t i o n o f R N A f r o m t h e 40 S, 60 S, a n d 75 S fractions o f rat skeletal m u s c l e ribosomes. R i b o s o m e s were dissociated in m e d i u m B a n d f r a c t i o n a t e d at 28 ° o n 1 0 - 3 0 % sucrose gradients, c o n t a i n i n g t h e s a m e buffer. T h e r i b o s o m e fractions were collected a n d t h e i r purity was d e t e r m i n e d (a-c) by analysis o n 1 5 - 3 0 % s u c r o s e g r a d i e n t s c o n t a i n i n g m e d i u m B w i t h o u t 2 - m e r c a p t o e t h a n o l . T h e a b s o r p t i o n at t h e t o p o f t h e s e g r a d i e n t s is d u e to 2 - m e r c a p t o e t h a n o l r e m a i n i n g f r o m the p r e p a r a t i v e m e d i u m . C e n t r i f u g a t i o n was at 28 ° for 40 m i n u t e s at 60,000 r p m in a SW 65 rotor (a-c). T h e s e d i m e n t a t i o n o f t h e R N A (d-f) isolated f r o m t h e fractions was d e t e r m i n e d as d e s c r i b e d in t h e text.
[43]
DISSOCIATION OF EUKARYOTIC CELL RIBOSOMES
(a)
"(b)
4O
0
T (e)
60 8O
80
(c)
I
4O
-(d)
423
60
(f)
6O
90
I
F~G. 4. Analysis o f rat liver ribosome s u b u n i t fractions. T h e fractions are f r o m the g r a d i e n t s h o w n in Fig. l a n d were collected at t h e points indicated by the arrows. Analysis was as described in t h e text.
is heavily contaminated with 40 S subunits (Fig. 4c), which is manifest by the presence of a large a m o u n t of 80 S ribosomes (the result of reassociation of subunits in m e d i u m A). T h e a m o u n t of contamination is decreased when the fractions are collected f u r t h e r from the center of rotation (Fig. 4 c-f); fractions e and f contain relatively small amounts of 40 S subunits (as reflected in the small amounts of 80 S and 105 S particles) and large numbers of 60 S subunits (i. e., 60 S and 90 S particles). T h e contamination of the 60 S peak is primarily the result of sedimentation of 40 S subunits as 55 S particles in m e d i u m B (Fig. 5). We do not know if the 55 S particle is a dimer or a more compact form of the 40 S subunit. If only that portion of the 60 S peak indicated by the bars (Fig. 1) is collected, it will contain 20-35% of the OD2~0 units applied to the gradient and will be contaminated with 5-10% of 40 S subunits. We have been unable to obtain mammalian 60 S subunits completely free of 40 S particles. Ribosomes Resistant to Dissociation. When ribosomes containing nascent protein labeled with radioactive amino acids in vitro are dissociated by
424
RIBOSOMES S T R U C T U R E AND F U N C T I O N
[43]
55S
O
¢,,
FIG. 5. S e d i m e n t a t i o n o f 40 S liver ribosomal s u b u n i t s in m e d i u m B. 40 S s u b u n i t s (fraction a o f Figs. 1 a n d 4) were dialyzed against m e d i u m B a n d t h e n layered o n t o 1 5 - 3 0 % linear s u c r o s e g r a d i e n t in m e d i u m B. C e n t r i f u g a t i o n was at 60,000 r p m for 40 m i n u t e s at 28 ° in a SW 65 rotor.
KCI, the greater part of the radioactivity remains attached to the 75 S component (Fig. 6). Thus it appears that ribosomes active in the synthesis of protein on endogenous messenger RNA are more resistant to dissociation. This conclusion is supported by the effect of preincubation of ribosomes to remove nascent protein and messenger RNA. Untreated rat liver ribosomes contain a greater proportion of monomers resistant to dissociation than do preparations from rat skeletal muscle (compare Figs. 2 and 7a). The proportion of the resistant 75 S particles is reduced by preincubation of the ribosomes with factors required for protein synthesis (Fig. 7b). If puromycin, which releases nascent peptide from ribosomes, is included in the preincubation medium, the particles completely dissociate at high concentrations of KC1 (Fig. 7c). Since the large proportion of 75 S particles that remain after dissociation of untreated liver ribosomes (Fig. 7a) results in low yields and causes inevitable contamination of the large subunit fraction, it is convenient to preincubate liver ribosomes with puromycin before preparation of the subunits. Preincubation can be carried out for 30 minutes at 37 ° in 60 ml of medium A containing 20 mM 2-mercaptoethanol, 5 mg of puromycin, approximately 6 0 0 r a g of liver supernatant protein, and 5000 OD~e0 units of ribosomes. After preincubation the ribosomes are reisolated in the customary way. It must be pointed out, however, that preincubafion with puromycin may modify the ribosomes, e. g., the
[43]
425
DISSOCIATION OF EUKARYOTIC CELL RIBOSOMES
(a) 80 m/W KCI
3000
0.6 2000 80S
0.4
I000 0.2
'~"
.£ E
tO
60S
I
(b) 880 mM KCI
C,
3000
0.6~ 2000
0.4
0,2
%
I000
I
I
FIG. 6. T h e presence o f nascent protein in the 75 S fraction o f rat skeletal muscle ribosomes dissociated by KCI at 28 °. Ribosomes ( 1 m g ribosomal RNA/ml) were incubated at 37 ° for 30 minutes in m e d i u m containing 50 m M Tris-HC1, p H 7.8, 80 m M KCI, 12.5 m M MgCIz, 5 m M ATP, 0.5 m M GTP, 1 m M phosphoenolpyruvate, 100 p,g/ml aminoacyl-tRNA containing 59,000 c p m [3H]phenylalanine. T h e reaction mixture (2 ml) was centrifuged on a 15-30% sucrose gradient containing: (a) m e d i u m A; (b) m e d i u m B without 2-mercaptoethanol. Centrifugation was at 28 ° for 5 hours at 22,500 r p m in a SW 25.1 rotor. T h e radioactivity o f the protein contained in l-ml fractions from the gradients was d e t e r m i n e d .
tendency to dimerizes and the magnesium dependency of polyphenylalanine synthesis.
426
RIBOSOMES STRUCTURE AND FUNCTION
0.4
(a) Untreated 75 S
(b) Preincubated 60 S
[43]
(c) Stripped
(puromycin) 60 S
0.3 0
60i/
0.2 O. I
40~ ~
F1c. 7. Effect o f preincubation and puromycin on the dissociation o f rat liver ribosomes. Rat liver ribosomes were (a) p r e p a r e d in the usual m a n n e r , (b) then incubated at 37 ° for 30 minutes in the m e d i u m for the assay o f protein synthesis, or (c) incubated in the m e d i u m for the assay o f protein synthesis containing in addition 5 0 / z g / m l puromycin; the p r e i n c u b a t e d ribosomes (b,c) were collected by centrifugation. T h e ribosomes (30/~g ribosomal RNA) were analyzed on 15-30% sucrose gradients containing m e d i u m B without 2-mercaptoethanol. Centrifugation was in a SW 65 rotor at 60,000 r p m for 30 minutes at 28 ° .
Reassociation of Ribosomal Subunits The 40 S and 60 S subunit fractions collected from sucrose gradients may be treated separately or mixed to prepare reassociated ribosomes. For reassociation, subunit fractions are combined in the ratio (60 S:40 S) of approximately 2.5:1 (ODin0); the proportions in which they are formed. 5 The separate subunit fractions or the combined subunits are then dialyzed overnight at 4 ° against medium A containing 20 mM
40S Subunit
60 SSubunil
Reossociated 80 S
0.5 40 ea
60 S
0.2 0.1
FIG. 8. T h e reassociation o f subunits o f rat skeletal muscle ribosomes. Separate and c o m b i n e d ribosomal subunits were dialyzed against m e d i u m A containing 20 m M 2m e r c a p t o e t h a n o l a n d analyzed on 15-30% sucrose gradients containing m e d i u m A. Centrifugation was at 60,000 r p m in a SW ,65 r o t o r for 30 minutes at 28 °.
[43]
427
DISSOCIATION OF EUKARYOTIC CELL RIBOSOMES
2-mercaptoethanol. After dialysis, the particles are analyzed by centrifugation on sucrose gradients containing medium A. The above procedure can lead to the complete reassociation of ribosomal subunits (Fig. 8). Consistently high levels of reassociation have been observed with rat, mouse, rabbit, yeast, and Tetrahymena pyriformis ribosomal subunits: wheat and pea have generally given lower yields (approximately 50%) apparently due to the greater lability of plant ribosomal subunits, particularly of the small subunit, s If ribosome subunit preparations from different sources are used, active hybrid 80 S particles may be obtained, n'7
Activity of the Subunits and of Reassociated Ribosomes When separate ribosomal subunit fractions or reassociated ribosomes are assayed for their ability to synthesize protein in a cell-free system very low activities are observed in the absence of added template RNA (see the table). The 40 S fraction is almost totally inactive even when assayed with polyuridylic acid, whereas the 60 S fraction has approxiPROTEIN SYNTHESIS BY SUBUNITS FROM RAT MUSCLE RIBOSOMES
Protein synthesis u (cpm/10/~g ribosomal RNA) Experimental conditions
Endogenous synthesis
Polyphenylalanine synthesis ~
527 307 0 16 18 18 377
4690 4593 178 1094 5415 4115 276
734 512 10 25 67
5910 4355 210 1465 6553
A. Assay after dialysis Untreated ribosomes KCl-treated ribosomes 40 S fraction 60 S fraction 40 S + 60 S mixed before dialysis 40 S + 60 S mixed after dialysis 75 S fraction
B. Direct assay Untreated ribosomes KCl-treated ribosomes 40 S fraction 60 S fraction 40S + 60S
~'The medium for the assay of protein synthesis contained (in 1 ml): 50 mM Tris.HC1 (pH 7.8), 88 mM KCI, 15 mM MgCI~, 10 mM 2-mercaptoethanol, 5 mM ATP, 0.4 mM GTP, 1 mM phosphoenolpyruvate, 10/~g of pyruvate kinase, 1.5-2% sucrose, 2 mg of muscle supernatant protein, and 100 ~g of aminoacyl-transfer RNA containing 30,000 cpm of [aH]phenylalanine. The amount of the ribosomal particles (ribosomal RNA) was 7.5-12.5 /zg, and the amount of polyuridylic acid added was 100 ~g. Incubation was at 37 ° for 30 minutes. Each 100 cpm in protein was the result of the incorporation of 2.3 x 10-13 moles of phenylalanine. bCorrected for endogenous incorporation of phenylalanine.
428
RIBOSOME STRUCTURE AND FUNCTION
[43]
mately 20% of the activity of the combined fractions (see the table), which accords with the presence of some 40 S particles in the 60 S fraction (Figs. 3 and 4). In the presence of polyuridylic acid the combined 60 S and 40 S fractions possess activity equal to, or exceeding that of the control ribosomes. The results are more consistent if the subunits are combined before dialysis, although there is activity if the subunits are combined after separate dialysis (part A of table). Activity is also restored if the subunit fractions are mixed, then assayed (without first being dialyzed) in medium whose ionic composition is adjusted to compensate for the excess of KC1 contained in the fractions (part B of table, direct assay). Rapid and specific reassociation of the subunits must occur in the reaction mixture. The direct assay, however, gives somewhat variable results and suffers the additional limitation that the products of reassociation cannot easily be analyzed on sucrose gradients. For these reasons ribosomal subunit fractions are generally dialyzed against medium A containing 20 mM 2-mercaptoethanol before assay of protein synthesis. Unlike the reassociated ribosomes which are inactive in the absence of added template RNA, the 75 S particles have almost the same endogenous activity as the control ribosomes; moreover, they are relatively unresponsive to polyuridylic acid (part A of table). These observations provide additional support for the view that the 75 S particles derive from ribosomes active in endogenous protein synthesis. The specific endogenous protein synthetic activity (cpm incorporated into protein per microgram of ribosomal RNA) of the 75 S fraction is not considerably greater than the original ribosomal preparation as would be expected if inactive particles have been removed by dissociation; thus, the experimental procedure must have caused some inactivation of the 75 S particles, although how much is not certain since the fraction does contain 60 S particles which do not have endogenous activity and would therefore lower the specific activity. The results suggest, however, that the dissociation procedure allows the separation of the ribosomes in the population that were active (the 75 S fraction) from subunits derived from ribosomes not carrying out endogenous protein synthesis, and this operation is potentially useful in the analysis of ribosome function. Remarks
The major difficulty associated with the procedure for the dissociation of ribosomes from eukaryotic cells, particularly those of mammals, is the contamination of the large subunit fraction by the small subunits.
[44]
ACTIVE SUBUNITS FROM LIVER RIBOSOMES
429
T h e contamination most likely arises f r o m the presence o f dimers or a m o r e compact f o r m o f the small subunit which cosediment with the large subunit in sucrose gradients. In addition, the 60 S subunit may be c o n t a m i n a t e d by 80 S ribosomes that are resistant to dissociation because they contain nascent peptide and messenger RNA; the latter source o f contamination can be minimized by p r e t r e a t i n g the ribosome with puromycin, although it must be realized that preincubation may slightly alter ribosome structure and f u n c t i o n / Despite the difficulty, the m e t h o d has already p r o v e d useful in locating a defect in the muscle ribosomes o f diabetic animals, 6 in determining that m R N A 11 and mRNA-directed tRNA binding 12is to the small subunit o f mammalian ribosomes, and in the investigation o f the interaction o f ribosomal subunits o f different species/ 11F.S. Rolleston, T. E. Martin, and I. G. Wool,Biochem.J. 117,899 (1970). ~.1.J. Castles and I. G. Wool,Biochemistry 9, 1909 (1970).
[44] The Dissociation of Rat Liver Ribosomes to Active Subunits by Urea By MARY L. PETERMANN W h e n rat liver ribosomes are dissociated by r e m o v i n g all their magnesium the 5 S RNA is detached and the subunits are inactive in an amino acid-incorporating system. 1 Active subunits can, however, be obtained by merely r e d u c i n g the ribosomal magnesium, then dissociating by t r e a t m e n t with u r e a / W i t h this p r o c e d u r e , precautions against d a m a g e by RNase are o f great importance. One must work rapidly, keep the material cold, and use an RNase inhibitor, such as bentonite. Bentonite. Bentonite (Fisher, USP) is equilibrated with 0.5 m M MgCI2, 1 m M potassium phosphate, p H 6.8, at 5 °, by s u s p e n d i n g 90 g in buffer in a blender, diluting the suspension to 2 liters, and stirring overnight. T h e suspension is c e n t r i f u g e d at 8000 g for 10 minutes. T h e pellets are discarded, and the s u p e r n a t a n t is used for the p r e p a r a t i o n o f "coarse" and "fine" fractions? T h e coarse bentonite is obtained by centrifuging the bentonite at 20,000 g for 15 minutes. T h e pellets are s u s p e n d e d in buffer with a pestle homogenizer, and the suspension is diluted to 1 liter, 1M. L. Petermann and A. Pavlovec,Biopolymers 7, 73 (1969). 2M. L. Petermann, A. Pavlovec,and I. B. Weinstein, Fed. Proc. Fed. Amer. Soc. Exp. Biol. 28, 725 (1969). 3M. L. Petermann and A. Pavlovec,J. Biol. Chem. 238, 3717 (1963).