- Email: [email protected]
[14] Isolation and purification of protein synthesis initiation factors from wheat germ
[14]
WHEAT GERM INITIATION FACTORS
193
on the same anion exchanger even at a lower salt concentration. A possible reason for this is that Co-eIF-2 may be complexed with another protein in the crude extract, and it is the complex that binds to DEAE-cellulose. A subsequent manipulation, such as elution from the DEAE-cellulose with 400 mM potassium chloride, precipitation with ammonium sulfate, or dialysis against 50 mM KC1, could disrupt the complex, thereby altering the behavior of the cofactor. A distinct advantage of this preparative procedure is that Co-eIF-2 is readily separated from eIF-2. The buffer A (400) eluate is virtually free of eIF-2. Occasionally some preparations have a high GTPindependent binding of Met-tRNAi which is probably due to nonspecific binding. A 2- to 3-fold stimulation of Met-tRNAi binding to elF-2 by Co-eIF-2 is routinely obtained although a maximum of about 5-fold has been observed in a few cases. Addition of Co-elF-2 to the standard reaction mixture for binding Met-tRNA~ to 40 S ribosomes does not cause an increase in the amount of 40 S complex formed. The mechanism of action of Co-eIF-2 is not known, but it would be attractive to assume that it forms a complex with eIF-2 and that this complex is the species which combines with GTP and Met-tRNAi. If this hypothesis is correct it should be possible to show a complete dependence upon Co-eIF-2 by very highly purified eIF-2. In the reticulocyte system .'° it has been shown that purified, but not crude, eIF-2 is stimulated by the cofactor. ~0 A. Dasgupta, A. Majumdar, A. D. George, and N. K. Gupta, Biochem. Biophys. Res. Cornmun. 71, 1234 (1976).
[14] I s o l a t i o n a n d P u r i f i c a t i o n o f P r o t e i n S y n t h e s i s Initiation Factors from Wheat Germ 1
By
B E N J. W A L T H A L L ,
LINDA L. SPREMULLI,
SANDRA R. LAX, and JOANNE M. RAVEL
Crude extracts of wheat germ are capable of translating mRN As from a variety of sources. '-4 Wheat germ extracts are easy to prepare, are low in nuclease activity, have low endogenous levels of mRNA, and translate exogenously supplied mRNAs with high efficiency and fidelity. Marcus This work was supported in part by U.S. Public Health Service Grant GM-18775. K. Marcu and B. Dudock, Nucl. Acids Res. 1, 1385 (1974). :~ B. E. Roberts and B. M. Patersofi, Proc. Natl. Acad. Sci. U.S.A. 70, 2330 (1973). 4 0 . E. Beck and H. G. G a s s e n , Biochern. Biophys. Res. Commun. 74, 16 (1977), and references cited in this paper.
METHODS 1N ENZYMOLOGY, VOL. LX
Copyright © 1979by AcademicPress. Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4
194
[14]
INITIATION OF PROTEIN SYNTHESIS SUPERNATANT
120 mM KCl POSTRIBOSOMAL
I 1AMMONIUM SULFATE I
m J
I
0-40%
40-60%
I
I DEAE- CELLULOSE l
I II 50ram KCl 120mMKCl
250mMKCl
IA
IB
I
.l
50mMKCI
IC
1
1
1
1
150mMKCI 250mMKCl
I ISEPHAOEXG-'501
I ISEPHAOEXG-'501 3B (elF- 3)
ID
250ram KCl
IE
120mMKCI 250mMKCl
2B
3A
120mMKCl
I
2A
(elF - 4 B)
~
I PHOSPHOCE''U'-OSEI
[ PHOSPHOCELLULOSEI I PHOSPHOCELLULOSEI
120mMKCI 250mMKCl
I
I OEAE-CEL'U'OSE
500mMKCl
2D
I
ISEPHAOEXG-,5Oj 3D (elF- 2)
FIG. 1. Purification scheme for wheat germ initiation factors.
and co-workers 5 have shown that the postribosomal supernatant fraction from wheat germ extracts can be resolved into four fractions that are required, in addition to elongation factors EF-1 and EF-2, for the in v i t r o translation of tobacco mosaic virus RNA. One of these fractions contains a factor that binds Met-tRNAf in the presence of GTP and appears to be analogous to elF-2" obtained from other eukaryotic cells. 7 A factor from wheat germ with similar properties has been purified and characterized by Treadwell and Robinson 8 and Spremulli e t a/. 9 The following sections describe the resolution of the 120 mM KCI postribosomal supernatant from wheat germ extracts into five fractions. These five fractions are required for maximal translation of yeast polysomal RNA in a cell-free system containing washed wheat germ ribosomes, wheat germ tRNA, and a level of initiation factor-deficient, postribosomal supernatant fraction sufficient to supply elongation factors and aminoacyl-tRNA synthetases. Factors presM. Giesen, R. Roman, S. N. Seal, and A. Marcus, J. Biol. Chem. 251, 6075 (1976). The nomenclature used for the initiation factors is that proposed at the International Symposium on Protein Synthesis held at Bethesda, Maryland, October, 1976; W. F. Anderson, L. Bosch, W. E. Cohn, H. Lodish, W. C. Merrick, H. Weissbach, H. G. Wittmann, and I. G. Wool, FEBS Lett. 76, 1 (1977). 7H. Weissbach and S. Ochoa, Annu. Rev. Biochem. 45, 191 (1976). s B. Treadwell and W. Robinson, Biochem. Biophys. Res. Commun. 65, 176 (1975). L. L. Spremulli, B. J. Walthall, S. R. Lax, and J. M. Ravel,Arch. Biochem. Biophys. 178, 565 (1977),
[14]
W H E A T GERM INITIATION FACTORS
195
ent in three of the fractions have been highly purified and appear to correspond to the mammalian initiation factors eIF-2, eIF-3, and elF-4B. The purification scheme for the wheat germ initiation factors is shown in Fig. 1. Materials Wheat germ is kindly supplied by Mr. J. Ryan of International Multifoods, Minneapolis, Minnesota, and is stored at - 2 0 ° until used. Yeast polysomal R N A is prepared essentially by the method of Gallis et al. ~o Wheat germ tRNA is purchased from Sigma. DEAE-cellulose (DE-52) and phosphocellulose (P-l l) are obtained from Whatman. Commonly used buffers are: Buffer A: 20 m M H E P E S - K O H , H pH 7.6; 5 m M Mg(OAc)2; 1 m M dithiothreitol; 10% glycerol; and KCI as indicated Buffer B: 20 m M H E P E S - K O H , pH 7.6; 0.1 m M EDTA; 1 m M dithiothreitol; 10% glycerol; and KCI as indicated. The millimolar concentration of KCI in the buffers is given following the buffer designation; e.g., buffer B-120 is buffer B containing 120 m M KCl. General Procedures All procedures are carried out at 0-4 ° unless otherwise indicated. All samples are divided into small and large atiquots, fast-frozen in a Dry I c e - i s o p r o p a n o l bath, and stored at --70 ° unless otherwise indicated. The small aliquots are used for assay purposes and discarded after being thawed once. The large aliquots are used for further purification steps. All column fractions are collected in siliconized tubes. Dialysis tubing is treated as previously described 1~ and stored at 4 °. All dilutions are made in plastic tubes and all samples are added with disposable plastic pipettes. Protein concentrations are determined by the method of Bensadoun and Weinstein 13 using bovine serum albumin as the standard. P r e p a r a t i o n of W h e a t G e r m Extracts a n d P o s t r i b o s o m a l S u p e r n a t a n t s Preparation o f the 120 m M KCI Postribosoma! Supernatant. Wheat germ (120 g) is reduced to a fine powder by grinding in a Waring blender at medium speed for four 15-sec intervals (shaken between grindings). The
~0B. M. Gallis, J. P. McDonnell,J. E. Hopper, and E. T. Young,Biochemistry 14, 1038(1975). 'f Abbreviations: HEPES, N-2-hydroxyethylpiperazine-N'-2-etbanesulfonic acid; EDTA, ethylenediarninetetraacetate; DEAE, diethylaminoetbyl; OAc,- acetyl; PPO, 2,5diphenyloxazole; SDS, sodium dodecyl sulfate. ,z p. McPhie, this series, Vol. 22, p, 23. ~'~A. Bensadoun and D. Weinstein, Anal. Biochem. 70, 241 (1976).
196
INITIATION OF PROTEIN SYNTHESIS
[14]
powdered wheat germ is mixed thoroughly with 200 ml of extraction buffer (20 mM H E P E S - K O H , pH 7.6, 1 mM Mg(OAc).~, 2 mM CaCl2, and 6 mM /3-mercaptoethanol) containing 120 mM KCI, transferred to centrifuge tubes, and centrifuged for 20 min at 15,000g. After centrifugation, the thick fatty layer on the top of the tubes is removed with a spatula and discarded; the supernatant solution is decanted and saved; and the pelleted material is discarded. The supernatant solution (about 170 ml containing 450-550 A 2~,} units/ml) is applied to a 1000-ml Sephadex G-25 column (5 × 50 cm) equilibrated in buffer A-120, and the column is developed with the same buffer at a flow rate of approximately 240 ml/hr. A single fraction of approximately 300 ml is collected starting with the emergence of material absorbing at 260 nm and ending when the absorbance drops below 90 A~0 units/ml. This fraction (containing approximately 130 A2,o units/ml) is clarified by centrifugation for 20 rain at 25,000 g and then is centrifuged for 3 hr at 170,000 g in a Beckman type 50.2 Ti rotor. The top three-quarters of the supernatant obtained from the 170,000 g centrifugation (designated the 120 mM KCI supernatant) is removed, immediately fractionated by ammonium sulfate precipitation as described below, and used as a source of initiation factors. The bottom one-fourth of the supernatant is discarded; the ribosomal pellet is suspended in buffer A-120 at a concentration of about 500 A.~0 units/ml and stored. Preparation o f the 20 m M K C l Postribosomal Supernatant. The 20 mM KCI postribosomal supernatant is prepared exactly as described above except that all the buffers contain 20 mM KC1 instead of 120 mM KCI. The top three-fourths of the supernatant from the 170,000 g centrifugation is removed and designated the 20 mM KC1 supernatant. It is divided into small and large aliquots and stored. The 20 mM KC1 supernatant has low initiation factor activity and can be used without further processing as a source of elongation factors and aminoacyl-tRNA synthetases in the polymerization assays described below for the various initiation factors (see Table I). The large aliquots are passed through a heparin-Sepharose column as described below. The 20 mM KC1 supernatant is passed through a column of heparinSepharose prepared as described by Goldstein. 14The KCl concentration of an aliquot of the 20 mM KCI supernatant is adjusted to 120 mM by the addition of 2.5 M KC1. Seventy milliliters of this material are applied to an 18-ml column of heparin-Sepharose (1. ! X 19 cm) equilibrated in buffer B-120. The column is developed with the same buffer, and fractions of 10 ml are collected at a flow rate of 18 ml/hr. Fractions 2 to 10 are pooled and the protein is precipitated by the addition of ammonium sulfate (80% of saturation). The precipitated protein is collected by centrifugation for 15 min at ~4A. A. Waldman,G. Marx,and J. Goldstein,Proc. Natl. Acad. Sci. U.S.A. 72, 2352(1975).
[14]
197
WHEAT GERM INITIATION FACTORS
18,000g, suspended in 20 ml of buffer A-20, and dialyzed overnight against buffer A-20. The dialyzed material (designated HS-20 mM KCI supernanant) is divided into small aliquots and stored. The HS-20 mM KCI supernatant has even lower initiation factor activity than the untreated 20 mM KCI supernatant and can also be used as a source of elongation factors and aminoacyl-tRNA synthetases in the polymerization assays (see Table V). Preparation of High-Salt Washed Ribosomes, The ribosomes obtained from extracts prepared in 120 mM KC1 are diluted with an equal volume of buffer A-1200 to give final concentrations of 650 mM KC1 and approximately 250 A 2~0units/ml of ribosomes. The ribosomes are allowed to stand in the high-salt buffer for 30 rain and are then collected by centrifugation for 4 hr at 140,000 g in a Beckman type 60 Ti rotor. The supernatant is discarded, and the ribosomes are suspended in buffer A-50 at a concentration'of about 500 A2~,~units/ml. The washed ribosomes are stored in small aliquots.
Preparation of 0--40% and 40--60% Ammonium Sulfate Fractions from the 120 mM KCI Postribosomal Supernatant. The 120 mM KCI supernatant (220 ml containing about 4 g of protein) is brought to 40% of saturation by the gradual addition of ammonium sulfate (22.6 g/100 ml). After stirring for 20 min, the precipitate is collected by centrifugation at 15,000 g for 15 rain. The supernatant is then brought to 60% of saturation by theaddition of 12.0 g of ammonium sulfate per 100 ml, and the precipitate is collected as described above. The two precipitates are each suspended in small amounts of buffer B-50 and are dialyzed against 100 volumes of the same buffer for 8 hr. After dialysis, the 0-40% fraction is clarified by centrifugation at 10,000 g for 10 rain. Both the 0-40 and 40-60% ammonium sulfate fractions are required to obtain maximal polymerization as shown in Table I. TABLE I COMPARISON OF THE AMOUNT OF POLYMERIZATION OBTAINED WITH 20 m M KCI SUPERNATANT AND 120 m M KCI SUPERNATANT AND AMMONIUM SULFATE FRACTIONATED 120 m M KCI SUPERNATANT Fraction added
Protein (mg)
[~4C]Leucine incorporated (pmol)
20 m M KCI s u p e r n a t a n t 20 m M KCI s u p e r n a t a n t 120 m M KCI s u p e r n a t a n t 0 - 4 0 % A m m o n i u m sulfate" 4 0 - 6 0 % A m m o n i u m sulfate" 0 - 4 0 % + 4 0 - 6 0 % A m m o n i u m sulfate"
0.3 1.0 1.0 0'.3 0.6 0.3 + 0.6
2.0 2.5 30.0 3.0 3.5 33.0
Derived from the 120 m M KCI s u p e r n a t a n t and a s s a y e d in the presence of 0.3 mg of 20 m M KC1 supernatant.
198
INITIATION OF PROTEIN SYNTHESIS
[14]
Fractionation of the 0-40% Ammonium Sulfate Fraction (Fig. 1)
Step 1. Separation of Fractions A and B by Chromatography on DEAE-Cellulose. The 0-40% ammonium sulfate fraction of the 120 m M KCl supernatant (15 ml containing 1000 mg of protein) is diluted to a protein concentration of about 45 mg/ml by the addition of buffer B-50 and is applied to a 70-ml DEAE-cellulose column (1.5 × 40 cm) equilibrated in buffer B-50. The column is washed with buffer B-50 until the absorbance at 280 nm is less than 0.2 and then developed successively with buffer B containing 120 and 250 mM KC1. Fractions of 7 ml are collected at a flow rate of about 50 ml/hr. The first 3 to 4 fractions containing protein eluted by 120 mM KCI are pooled and designated fraction 1A. The first 3 or 4 fractions containing protein eluted by 250 mM KC1 are pooled and designated fraction lB. In some cases the first fraction containing protein eluted by 250 mM KCI is not included because it contains detectable amounts of the activity found in fraction IA. Step 2A. Chromatography of Fraction lA on Phosphocellulose. Fraction IA (28 ml containing 160 mg of protein) is applied to a 25-m1 phosphocellulose column (1.1 X 27 cm) equilibrated in buffer B-120. The column is washed with the same buffer until the absorbance at 280 nm is less than 0.2 and the activity is eluted from the column with buffer B-250. Fractions of 2.5 ml are collected at a flow rate of 30 ml/hr. The active fractions (generally 3 or 4) are pooled and designated fraction 2A. Step 3A. Chromatography of Fraction 2A on Sephadex G-150. A portion of fraction 2A (3 ml containing 6 mg of protein) is concentrated to I ml by dialysis overnight against buffer B-100 containing 50% glycerol and is applied to a 100-ml Sephadex G-150 column (1.5 X 60 cm) equilibrated in buffer B-100. The column is developed with the same buffer and fractions of 2 ml are collected at a flow rate of 20 ml/hr. The activity elutes from the column at or slightly behind the void volume. It is important that a protein, such as bovine serum albumin, be passed through the Sephadex G-150 column prior to application of the sample; otherwise, poor recoveries of the fraction A activity are obtained. Step 2B. Chromatography of Fraction IB on Phosphocellulose. Fraction 1B (21 ml containing 205 mg of protein) is diluted with buffer B-0 to reduce the concentration of KCI to 150 mM and is applied to a 34-ml phosphocellulose column (1.1 X 36 cm) equilibrated in buffer B-150. The column is washed with the same buffer until the absorbance at 280 nm is less that 0.2, and the activity is eluted with buffer B containing 250 mM KCI. Fractions of 3.5 ml are collected at a flow rate of approximately 20 ml/hr. The active fractions (generally 3 or 4) are pooled and designated fraction 2B. Step 3B. Chromatography of Fraction 2B on Sephadex G-150. A pot-
[14]
W H E A T GERM INITIATION FACTORS
199
TABLE II PURIFICATIONOF FACTORSA AND B FROMTHE 0--40% AMMONIUMSULFATEFRACTIONa
Procedure
Total Total protein u n i t s (mg) (× lO a)
Specific activity (units/mg)
Yield (%)
Purification (fold)
Factor A 1A DEAL-cellulose (120 mM KC1) 2A Phosphocellulose (250 mM KC1) 3A SephadexG-150
160
21
130
100
--
10
13
1300
62
10
5
4200
24
32
205
53
260
100
--
10
26
2600
49
10
11
9200
21
35
1,2
Factor B IB DEAL-cellulose (250 mM KCI) 2B Phosphocellulose (250 mM KCI) 3B Sephadex G-150
1.2
" The 0-40% ammonium sulfate fraction obtained from 120g of wheat germ contains 1000 mg of protein,
tion of fraction 2B (3 ml containing 6 mg of protein) is concentrated to 1 ml by dialysis overnight against buffer B-100 containing 50% glycerol and is applied to a 100-ml Sephadex G-150 column (1.5 × 60 cm) equilibrated in buffer B-100. The column is developed with the same buffer, and fractions of 2 ml are collected at a flow rate of 20 ml/hr, The activity elutes from the column at the void volume. The active fractions are either stored or dialyzed overnight against buffer B- 100 containing 50% glycerol and stored at - 15°. A summary of the purification of fractions A and B is given in Table lI. F r a c t i o n a t i o n of the 4 0 - 6 0 % A m m o n i u m Sulfate F r a c t i o n (Fig. 1)
Step I. Separation of Fractions C, D, and E by Chromatography on DEAL-Cellulose. The 40-60% ammonium sulfate fraction of the 120 m M KC1 supernatant (25 ml containing 1800 mg of protein) is diluted to a protein concentration of about 35 mg/ml by the addition of buffer B-50 and is applied to a 60-ml DEAL-cellulose column (1. I × 60 cm) equilibrated in buffer B-50. The column is washed with the same buffer and is then developed successively with buffer B containing 120 and 250 m M KCI. Fractions of 6 ml are collected at a flow rate of 50 ml/hr. The first 8-10 fractions containing protein that is not retained by the column in 50 m M KC1 are pooled, concentrated about 3-fold by ultrafiltration with an Ami-
200
[14|
INITIATION OF PROTEIN SYNTHESIS
con PM-10 filter, and stored in small aliquots. This fraction is designated I C. All the fractions containing protein eluted by 120 mM KCI (generally 5-8 fractions) are pooled and designated fraction 1D. It is important that all the fractions containing significant amounts of protein be pooled, since there is some indication that two factors may be eluted by 120 mM KC1, one of which elutes behind the major peak of protein. The first 3 or 4 fractions containing protein eluted by 250 m M KCI are pooled, concentrated about 3-fold by ultrafiltration with an Amicon PM-10 filter, and stored in small aliquots. This fraction is designated IE. The separation of fractions C, D, and E by chromatography on DEAE-cellulose is summarized in Table III. Step 2D. Chromatography of Fraction 1D on Phosphocellulose. Two preparations of fraction ID obtained from two DEAE-cellulose columns are pooled (70 ml containing 440 mg of protein) and applied to a 40-ml phosphocellulose column (1.5 x 25 cm) equilibrated in buffer B-120. The column is washed with the same buffer and is then developed successively with buffer B containing 250 and 500 mM KCI. Fractions of 4 ml are collected at a flow rate of about 25 ml/hr. The activity is eluted by 500 mM KC1 and most of the activity is recovered in one or two fractions. This fraction is designated 2D. Step 3D. Chromatography of Fraction 2D on Sephadex G-150. Three milliliters of fraction 2D are concentrated by dialysis overnight against B-100 containing 50% glycerol. This material (1.0 ml containing 6 mg of protein) is applied to a 100-ml Sephadex G-150 column (1.5 X 60 cm) equilibrated in buffer B-100 and the column is developed with the same buffer. Fractions of 2 ml are collected at a flow rate of 20 ml/hr. The activity elutes from the column at 1.2-1.3 times the void volume. The purification of the factor in fraction D, which forms a ternary complex with Met-tRNAf and GTP, is summarized in Table IV.
TABLE III FRACTIONATION OF THE 40--60% AMMONIUM SULFATE FRACTION ON DEAE-CELLULOSE"
Fraction
KCI (raM)
Total protein (rag)
Total units ( × 10-'~)
Specific activity (units/rag)
1C ID IE
50 120 250
332 220 206
75 6 33
226 27 160
" The 40-60% ammonium sulfate fraction (1.8 g of protein) obtained from !20 g of wheat germ is applied to the DEAE-cellulose column.
[14]
201
W H E A T GERM I N I T I A T I O N FACTORS
E
E
¢q
O
r--
'5
~X
g~ Z ©
E E
>7-
I
¢q Z ©
Y:
r~
¢q
,~
E
C)
r~
Z
O
r~
r~
~2
202
I N I T I A T I O N OF P R O T E I N S Y N T H E S I S
[14]
Assay Methods
Polymerization Assay. The standard polymerization assay system contains in a total volume of 0.1 or 0.15 ml: 24 mM H E P E S - K O H , pH 7.6, 2.4 mM dithiothreitol, 0. ! mM spermine, 3.5 mM Mg(OAc).~, 85-90 mM KCI, 3-9% glycerol, 18 ~ [~4C]leucine (approximately 160 cpm/pmol), 50/All of the other 19 amino acids, 1 mM ATP (neutralized with KOH), 0.2 mM GTP, 7.8 mM creadne phosphate, 3 p+g of creatine phosphokinase, 0.75 A.,~0 unit of wheat germ tRNA, 1.2 A.~0 units of yeast polysomal RNA, 1.8-2.5A260 units of washed wheat germ ribosomes, 300 p,g of 20 mM KC1 postribosomal supernatant and fractions derived from the 120 mM KCI postribosomal supernatant as indicated below. The reaction mixture is incubated for 30 rain at 25°. The reaction is terminated by the addition of 3 mi of 5% trichloroacetic acid and heated for 10 min at 90°. The heated mixture is passed through a Millipore filter (type HA, 0.45/zm) and the filter is washed with two 3-ml portions of 1% trichloroacetic acid and one 3-ml portion of water. The filter is dried by heating at 100° for 7 min, placed in a vial containing 15 ml of scintillation fluid (5 g of PPO per liter of toluene), and the radioactivity is measured in a liquid scintillation counter. A unit is defined as ! pmol of [~4C]leucine incorporated into polypeptide under the conditions described. To assay for the various activities, the standard reaction mixture is supplemented as follows: Fraction A activity: 450/zg of the 40-60% ammonium sulfate fraction and 10/zg of 2B Fraction B activity: 450/~g of the 40-60% ammonium sulfate fraction and 14/zg of 2A Fraction C activity: 14 ~g of 2A, l0/zg of2B, 6 p,g of 2D, and 250/zg of 1E Fraction D activity: 14 ~g of 2A, 10 ~g of 2B, 160/zg of 1C, and 250/zg of 1E Fraction E activity: 14/.~gof2A, 10p+gof2B, 160~g of IC, and 6p+g of 2D Incorporation of [l+C]leucine into polypeptide is linear up to 1.5 hr. The amount of polymerization obtained is reduced about 2-fold when spermine is omitted and is highly dependent upon the concentrations of KCI, Mg '~+, and glycerol. The KC1 concentration is carefully maintained between 87 and 93 mM, taking into consideration the KCI contributed by all samples added to the assay. The optimum Mg 2+ concentration is between 3 and 4 mM. High concentrations of glycerol are inhibitory, and the total glycerol concentration in the assay is therefore kept below 9%. The amount of 20 mM KC1 supernatant used in the assay to supply elongation factors and
[14]
203
W H E A T GERM I N I T I A T I O N FACTORS
aminoacyl-tRNA synthetases is based on the amount required to support near maximal poly(U)-directed polyphenylalanine synthesis. Ternary Complex Assay. The standard ternary complex (elF-2-MettRNAf GTP) assay system contains in a total volume of 0.1 ml: 24 mM HEPES-KOH, pH 7.6, 100 mM KC1, 2.4 mM dithiothreitol, 0.02 mM GTP, 10-15 pmol of yeast [3'~S]Met-tRNAf9 (500-5000 cpm/pmol), and fraction D. The reaction mixture is incubated for 5 min at 25°. The reaction is terminated by the addition of 2 ml of cold buffer containing 20 mM Tris. HCI, pH 7.7, and 100 mM KC1, and is passed through a Millipore filter (type HA, 0.45/zm). The filter is washed with two 2-mi portions of the same buffer and dried, and the radioactivity is measured as described above. A unit is defined as 1 pmol of [35S]Met-tRNAf retained on the filter under the conditions described. Properties of the Purified Factors The dependence of polypeptide synthesis on the various fractions obtained from the 120 mM KC1 supernatant is shown in Table V. As shown in TABLE V DEPENDENCE OF POLYPEPTIDE SYNTHESIS ON VARIOUS FRACTIONS OBTAINED FROM THE 120 m M KC1 POSTRIBOSOMAL SUPERNATANT ° In the presence of untreated 20 m M KCI supernatant
In the presence of heparin-Sepharose-treated 20 m M KCI supernatant
Fraction omitted
['4C]Leucine incorporated (pmol)
Decrease (fold)
['4C]Leucine incorporated (pmol)
Decrease (fold)
None A B C D E
17 9 4 6 9 2
-1.9 4.3 2.8 1.9 8.5
14 2 1 5 4 2
-7.0 14.0 2.8 3.5 7.0
" T h e complete standard reaction mixture is s u p p l e m e n t e d with preparations of the variotis fractions as follows: 14 pg of 2A, 10/xg of 2B, 160/xg of IC, 7/xg of 2D, and 250/zg of 1E, and 0.32 mg of 20 m M KCI s u p e r n a t a n t or 0.43 mg of h e p a r i n - S e p h a r o s e - t r e a t e d 20 m M KCI supernatant as indicated. The values given above are corrected for the a m o u n t of polymerization obtained w h e n all the fractions are omitted (2 pmol with the untreated supernatant and 1 pmol with the h e p a r i n - S e p h a r o s e - t r e a t e d supernatant). W h e n all five fractions are a s s a y e d without a n y s u p e r n a t a n t being present, 5 pmol of polymerization are observed.
204
INITIATION
OF PROTEIN
SYNTHESIS
[15]
the table, a greater dependence on the fractions is obtained when heparin-Sepharose-treated 20 mM KC1 supernatant is used as a source of elongation factors and aminoacyi-tRNA synthetases rather than the untreated supernatant. Factor in Fraction A. Electrophoresis of fraction 3A (Sephadex G-150 fraction) on SDS polyacrylamide gels shows that this fraction contains two major species of protein, one with a molecular weight of approximately 80,000 and one greater than 100,000. Correlation of the activity with the intensity of the protein bands present in consecutive fractions obtained from Sephadex G-150 indicates that the active component is the 80,000 molecular weight species, strongly suggesting that this factor is comparable to elF-4B from rabbit reticulocytes. Benne et al. J5 have shown that elF-4B from rabbit reticulocytes is in the 0-40% ammonium sulfate fraction and is eluted from DEAE-cellulose and phosphocellulose at comparable salt concentrations. Factor in Fraction B. Glycerol density gradient centrifugation of fraction 2B indicates that the active component in this fraction has an s value of approximately 17 S. SDS polyacrylamide gel electrophoresis of fraction 3B shows 9-11 bands ranging in molecular weight from about 35,000 to 160,000. These data indicate that the active component in this fraction corresponds to elF-3 from mammalian cells. Factor in Fraction D. As previously reported, fraction D contains elF-2, the factor that forms a ternary complex with Met-tRNAe and GTP. Factors in Fractions C and E. These fractions have not been purified sufficiently for identification of the active components. Each of these fractions may contain more than one initiation factor and probably contains some elongation factors and aminoacyl-tRNA synthetases. ~ R. B e n n e , M. L u e d i , a n d J. W. B. H e r s h e y , J. Biol. Chem. 252, 5798 (1977).
[15] P l e i o t r o p i c E f f e c t s o f I n i t i a t i o n F a c t o r I F - 1 By H. O. VOORMA, R. BENNE, N. NAAKTGEBOREN,
and G. A. J. M. VAN DER HOESTAD Initiation factor IF-1 has a considerable effect on several steps in the initiation of protein synthesis in Escherichia coll. Since the molecular weight is only 9000 it is reasonable to assume that these different effects are caused by one basic action of IF-I which influences the reaction rates. The different effects of IF-1 are as follows: 1. Increase of the binding of IF-2 and fMet-tRNA to the 30 S subunit 2. Increase of the rate of recycling of IF-2
METHODS IN ENZYMOLOGY, VOL. LX
Copyright © 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4
WHEAT GERM INITIATION FACTORS
193
on the same anion exchanger even at a lower salt concentration. A possible reason for this is that Co-eIF-2 may be complexed with another protein in the crude extract, and it is the complex that binds to DEAE-cellulose. A subsequent manipulation, such as elution from the DEAE-cellulose with 400 mM potassium chloride, precipitation with ammonium sulfate, or dialysis against 50 mM KC1, could disrupt the complex, thereby altering the behavior of the cofactor. A distinct advantage of this preparative procedure is that Co-eIF-2 is readily separated from eIF-2. The buffer A (400) eluate is virtually free of eIF-2. Occasionally some preparations have a high GTPindependent binding of Met-tRNAi which is probably due to nonspecific binding. A 2- to 3-fold stimulation of Met-tRNAi binding to elF-2 by Co-eIF-2 is routinely obtained although a maximum of about 5-fold has been observed in a few cases. Addition of Co-elF-2 to the standard reaction mixture for binding Met-tRNA~ to 40 S ribosomes does not cause an increase in the amount of 40 S complex formed. The mechanism of action of Co-eIF-2 is not known, but it would be attractive to assume that it forms a complex with eIF-2 and that this complex is the species which combines with GTP and Met-tRNAi. If this hypothesis is correct it should be possible to show a complete dependence upon Co-eIF-2 by very highly purified eIF-2. In the reticulocyte system .'° it has been shown that purified, but not crude, eIF-2 is stimulated by the cofactor. ~0 A. Dasgupta, A. Majumdar, A. D. George, and N. K. Gupta, Biochem. Biophys. Res. Cornmun. 71, 1234 (1976).
[14] I s o l a t i o n a n d P u r i f i c a t i o n o f P r o t e i n S y n t h e s i s Initiation Factors from Wheat Germ 1
By
B E N J. W A L T H A L L ,
LINDA L. SPREMULLI,
SANDRA R. LAX, and JOANNE M. RAVEL
Crude extracts of wheat germ are capable of translating mRN As from a variety of sources. '-4 Wheat germ extracts are easy to prepare, are low in nuclease activity, have low endogenous levels of mRNA, and translate exogenously supplied mRNAs with high efficiency and fidelity. Marcus This work was supported in part by U.S. Public Health Service Grant GM-18775. K. Marcu and B. Dudock, Nucl. Acids Res. 1, 1385 (1974). :~ B. E. Roberts and B. M. Patersofi, Proc. Natl. Acad. Sci. U.S.A. 70, 2330 (1973). 4 0 . E. Beck and H. G. G a s s e n , Biochern. Biophys. Res. Commun. 74, 16 (1977), and references cited in this paper.
METHODS 1N ENZYMOLOGY, VOL. LX
Copyright © 1979by AcademicPress. Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4
194
[14]
INITIATION OF PROTEIN SYNTHESIS SUPERNATANT
120 mM KCl POSTRIBOSOMAL
I 1AMMONIUM SULFATE I
m J
I
0-40%
40-60%
I
I DEAE- CELLULOSE l
I II 50ram KCl 120mMKCl
250mMKCl
IA
IB
I
.l
50mMKCI
IC
1
1
1
1
150mMKCI 250mMKCl
I ISEPHAOEXG-'501
I ISEPHAOEXG-'501 3B (elF- 3)
ID
250ram KCl
IE
120mMKCI 250mMKCl
2B
3A
120mMKCl
I
2A
(elF - 4 B)
~
I PHOSPHOCE''U'-OSEI
[ PHOSPHOCELLULOSEI I PHOSPHOCELLULOSEI
120mMKCI 250mMKCl
I
I OEAE-CEL'U'OSE
500mMKCl
2D
I
ISEPHAOEXG-,5Oj 3D (elF- 2)
FIG. 1. Purification scheme for wheat germ initiation factors.
and co-workers 5 have shown that the postribosomal supernatant fraction from wheat germ extracts can be resolved into four fractions that are required, in addition to elongation factors EF-1 and EF-2, for the in v i t r o translation of tobacco mosaic virus RNA. One of these fractions contains a factor that binds Met-tRNAf in the presence of GTP and appears to be analogous to elF-2" obtained from other eukaryotic cells. 7 A factor from wheat germ with similar properties has been purified and characterized by Treadwell and Robinson 8 and Spremulli e t a/. 9 The following sections describe the resolution of the 120 mM KCI postribosomal supernatant from wheat germ extracts into five fractions. These five fractions are required for maximal translation of yeast polysomal RNA in a cell-free system containing washed wheat germ ribosomes, wheat germ tRNA, and a level of initiation factor-deficient, postribosomal supernatant fraction sufficient to supply elongation factors and aminoacyl-tRNA synthetases. Factors presM. Giesen, R. Roman, S. N. Seal, and A. Marcus, J. Biol. Chem. 251, 6075 (1976). The nomenclature used for the initiation factors is that proposed at the International Symposium on Protein Synthesis held at Bethesda, Maryland, October, 1976; W. F. Anderson, L. Bosch, W. E. Cohn, H. Lodish, W. C. Merrick, H. Weissbach, H. G. Wittmann, and I. G. Wool, FEBS Lett. 76, 1 (1977). 7H. Weissbach and S. Ochoa, Annu. Rev. Biochem. 45, 191 (1976). s B. Treadwell and W. Robinson, Biochem. Biophys. Res. Commun. 65, 176 (1975). L. L. Spremulli, B. J. Walthall, S. R. Lax, and J. M. Ravel,Arch. Biochem. Biophys. 178, 565 (1977),
[14]
W H E A T GERM INITIATION FACTORS
195
ent in three of the fractions have been highly purified and appear to correspond to the mammalian initiation factors eIF-2, eIF-3, and elF-4B. The purification scheme for the wheat germ initiation factors is shown in Fig. 1. Materials Wheat germ is kindly supplied by Mr. J. Ryan of International Multifoods, Minneapolis, Minnesota, and is stored at - 2 0 ° until used. Yeast polysomal R N A is prepared essentially by the method of Gallis et al. ~o Wheat germ tRNA is purchased from Sigma. DEAE-cellulose (DE-52) and phosphocellulose (P-l l) are obtained from Whatman. Commonly used buffers are: Buffer A: 20 m M H E P E S - K O H , H pH 7.6; 5 m M Mg(OAc)2; 1 m M dithiothreitol; 10% glycerol; and KCI as indicated Buffer B: 20 m M H E P E S - K O H , pH 7.6; 0.1 m M EDTA; 1 m M dithiothreitol; 10% glycerol; and KCI as indicated. The millimolar concentration of KCI in the buffers is given following the buffer designation; e.g., buffer B-120 is buffer B containing 120 m M KCl. General Procedures All procedures are carried out at 0-4 ° unless otherwise indicated. All samples are divided into small and large atiquots, fast-frozen in a Dry I c e - i s o p r o p a n o l bath, and stored at --70 ° unless otherwise indicated. The small aliquots are used for assay purposes and discarded after being thawed once. The large aliquots are used for further purification steps. All column fractions are collected in siliconized tubes. Dialysis tubing is treated as previously described 1~ and stored at 4 °. All dilutions are made in plastic tubes and all samples are added with disposable plastic pipettes. Protein concentrations are determined by the method of Bensadoun and Weinstein 13 using bovine serum albumin as the standard. P r e p a r a t i o n of W h e a t G e r m Extracts a n d P o s t r i b o s o m a l S u p e r n a t a n t s Preparation o f the 120 m M KCI Postribosoma! Supernatant. Wheat germ (120 g) is reduced to a fine powder by grinding in a Waring blender at medium speed for four 15-sec intervals (shaken between grindings). The
~0B. M. Gallis, J. P. McDonnell,J. E. Hopper, and E. T. Young,Biochemistry 14, 1038(1975). 'f Abbreviations: HEPES, N-2-hydroxyethylpiperazine-N'-2-etbanesulfonic acid; EDTA, ethylenediarninetetraacetate; DEAE, diethylaminoetbyl; OAc,- acetyl; PPO, 2,5diphenyloxazole; SDS, sodium dodecyl sulfate. ,z p. McPhie, this series, Vol. 22, p, 23. ~'~A. Bensadoun and D. Weinstein, Anal. Biochem. 70, 241 (1976).
196
INITIATION OF PROTEIN SYNTHESIS
[14]
powdered wheat germ is mixed thoroughly with 200 ml of extraction buffer (20 mM H E P E S - K O H , pH 7.6, 1 mM Mg(OAc).~, 2 mM CaCl2, and 6 mM /3-mercaptoethanol) containing 120 mM KCI, transferred to centrifuge tubes, and centrifuged for 20 min at 15,000g. After centrifugation, the thick fatty layer on the top of the tubes is removed with a spatula and discarded; the supernatant solution is decanted and saved; and the pelleted material is discarded. The supernatant solution (about 170 ml containing 450-550 A 2~,} units/ml) is applied to a 1000-ml Sephadex G-25 column (5 × 50 cm) equilibrated in buffer A-120, and the column is developed with the same buffer at a flow rate of approximately 240 ml/hr. A single fraction of approximately 300 ml is collected starting with the emergence of material absorbing at 260 nm and ending when the absorbance drops below 90 A~0 units/ml. This fraction (containing approximately 130 A2,o units/ml) is clarified by centrifugation for 20 rain at 25,000 g and then is centrifuged for 3 hr at 170,000 g in a Beckman type 50.2 Ti rotor. The top three-quarters of the supernatant obtained from the 170,000 g centrifugation (designated the 120 mM KCI supernatant) is removed, immediately fractionated by ammonium sulfate precipitation as described below, and used as a source of initiation factors. The bottom one-fourth of the supernatant is discarded; the ribosomal pellet is suspended in buffer A-120 at a concentration of about 500 A.~0 units/ml and stored. Preparation o f the 20 m M K C l Postribosomal Supernatant. The 20 mM KCI postribosomal supernatant is prepared exactly as described above except that all the buffers contain 20 mM KC1 instead of 120 mM KCI. The top three-fourths of the supernatant from the 170,000 g centrifugation is removed and designated the 20 mM KC1 supernatant. It is divided into small and large aliquots and stored. The 20 mM KC1 supernatant has low initiation factor activity and can be used without further processing as a source of elongation factors and aminoacyl-tRNA synthetases in the polymerization assays described below for the various initiation factors (see Table I). The large aliquots are passed through a heparin-Sepharose column as described below. The 20 mM KC1 supernatant is passed through a column of heparinSepharose prepared as described by Goldstein. 14The KCl concentration of an aliquot of the 20 mM KCI supernatant is adjusted to 120 mM by the addition of 2.5 M KC1. Seventy milliliters of this material are applied to an 18-ml column of heparin-Sepharose (1. ! X 19 cm) equilibrated in buffer B-120. The column is developed with the same buffer, and fractions of 10 ml are collected at a flow rate of 18 ml/hr. Fractions 2 to 10 are pooled and the protein is precipitated by the addition of ammonium sulfate (80% of saturation). The precipitated protein is collected by centrifugation for 15 min at ~4A. A. Waldman,G. Marx,and J. Goldstein,Proc. Natl. Acad. Sci. U.S.A. 72, 2352(1975).
[14]
197
WHEAT GERM INITIATION FACTORS
18,000g, suspended in 20 ml of buffer A-20, and dialyzed overnight against buffer A-20. The dialyzed material (designated HS-20 mM KCI supernanant) is divided into small aliquots and stored. The HS-20 mM KCI supernatant has even lower initiation factor activity than the untreated 20 mM KCI supernatant and can also be used as a source of elongation factors and aminoacyl-tRNA synthetases in the polymerization assays (see Table V). Preparation of High-Salt Washed Ribosomes, The ribosomes obtained from extracts prepared in 120 mM KC1 are diluted with an equal volume of buffer A-1200 to give final concentrations of 650 mM KC1 and approximately 250 A 2~0units/ml of ribosomes. The ribosomes are allowed to stand in the high-salt buffer for 30 rain and are then collected by centrifugation for 4 hr at 140,000 g in a Beckman type 60 Ti rotor. The supernatant is discarded, and the ribosomes are suspended in buffer A-50 at a concentration'of about 500 A2~,~units/ml. The washed ribosomes are stored in small aliquots.
Preparation of 0--40% and 40--60% Ammonium Sulfate Fractions from the 120 mM KCI Postribosomal Supernatant. The 120 mM KCI supernatant (220 ml containing about 4 g of protein) is brought to 40% of saturation by the gradual addition of ammonium sulfate (22.6 g/100 ml). After stirring for 20 min, the precipitate is collected by centrifugation at 15,000 g for 15 rain. The supernatant is then brought to 60% of saturation by theaddition of 12.0 g of ammonium sulfate per 100 ml, and the precipitate is collected as described above. The two precipitates are each suspended in small amounts of buffer B-50 and are dialyzed against 100 volumes of the same buffer for 8 hr. After dialysis, the 0-40% fraction is clarified by centrifugation at 10,000 g for 10 rain. Both the 0-40 and 40-60% ammonium sulfate fractions are required to obtain maximal polymerization as shown in Table I. TABLE I COMPARISON OF THE AMOUNT OF POLYMERIZATION OBTAINED WITH 20 m M KCI SUPERNATANT AND 120 m M KCI SUPERNATANT AND AMMONIUM SULFATE FRACTIONATED 120 m M KCI SUPERNATANT Fraction added
Protein (mg)
[~4C]Leucine incorporated (pmol)
20 m M KCI s u p e r n a t a n t 20 m M KCI s u p e r n a t a n t 120 m M KCI s u p e r n a t a n t 0 - 4 0 % A m m o n i u m sulfate" 4 0 - 6 0 % A m m o n i u m sulfate" 0 - 4 0 % + 4 0 - 6 0 % A m m o n i u m sulfate"
0.3 1.0 1.0 0'.3 0.6 0.3 + 0.6
2.0 2.5 30.0 3.0 3.5 33.0
Derived from the 120 m M KCI s u p e r n a t a n t and a s s a y e d in the presence of 0.3 mg of 20 m M KC1 supernatant.
198
INITIATION OF PROTEIN SYNTHESIS
[14]
Fractionation of the 0-40% Ammonium Sulfate Fraction (Fig. 1)
Step 1. Separation of Fractions A and B by Chromatography on DEAE-Cellulose. The 0-40% ammonium sulfate fraction of the 120 m M KCl supernatant (15 ml containing 1000 mg of protein) is diluted to a protein concentration of about 45 mg/ml by the addition of buffer B-50 and is applied to a 70-ml DEAE-cellulose column (1.5 × 40 cm) equilibrated in buffer B-50. The column is washed with buffer B-50 until the absorbance at 280 nm is less than 0.2 and then developed successively with buffer B containing 120 and 250 mM KC1. Fractions of 7 ml are collected at a flow rate of about 50 ml/hr. The first 3 to 4 fractions containing protein eluted by 120 mM KCI are pooled and designated fraction 1A. The first 3 or 4 fractions containing protein eluted by 250 mM KC1 are pooled and designated fraction lB. In some cases the first fraction containing protein eluted by 250 mM KCI is not included because it contains detectable amounts of the activity found in fraction IA. Step 2A. Chromatography of Fraction lA on Phosphocellulose. Fraction IA (28 ml containing 160 mg of protein) is applied to a 25-m1 phosphocellulose column (1.1 X 27 cm) equilibrated in buffer B-120. The column is washed with the same buffer until the absorbance at 280 nm is less than 0.2 and the activity is eluted from the column with buffer B-250. Fractions of 2.5 ml are collected at a flow rate of 30 ml/hr. The active fractions (generally 3 or 4) are pooled and designated fraction 2A. Step 3A. Chromatography of Fraction 2A on Sephadex G-150. A portion of fraction 2A (3 ml containing 6 mg of protein) is concentrated to I ml by dialysis overnight against buffer B-100 containing 50% glycerol and is applied to a 100-ml Sephadex G-150 column (1.5 X 60 cm) equilibrated in buffer B-100. The column is developed with the same buffer and fractions of 2 ml are collected at a flow rate of 20 ml/hr. The activity elutes from the column at or slightly behind the void volume. It is important that a protein, such as bovine serum albumin, be passed through the Sephadex G-150 column prior to application of the sample; otherwise, poor recoveries of the fraction A activity are obtained. Step 2B. Chromatography of Fraction IB on Phosphocellulose. Fraction 1B (21 ml containing 205 mg of protein) is diluted with buffer B-0 to reduce the concentration of KCI to 150 mM and is applied to a 34-ml phosphocellulose column (1.1 X 36 cm) equilibrated in buffer B-150. The column is washed with the same buffer until the absorbance at 280 nm is less that 0.2, and the activity is eluted with buffer B containing 250 mM KCI. Fractions of 3.5 ml are collected at a flow rate of approximately 20 ml/hr. The active fractions (generally 3 or 4) are pooled and designated fraction 2B. Step 3B. Chromatography of Fraction 2B on Sephadex G-150. A pot-
[14]
W H E A T GERM INITIATION FACTORS
199
TABLE II PURIFICATIONOF FACTORSA AND B FROMTHE 0--40% AMMONIUMSULFATEFRACTIONa
Procedure
Total Total protein u n i t s (mg) (× lO a)
Specific activity (units/mg)
Yield (%)
Purification (fold)
Factor A 1A DEAL-cellulose (120 mM KC1) 2A Phosphocellulose (250 mM KC1) 3A SephadexG-150
160
21
130
100
--
10
13
1300
62
10
5
4200
24
32
205
53
260
100
--
10
26
2600
49
10
11
9200
21
35
1,2
Factor B IB DEAL-cellulose (250 mM KCI) 2B Phosphocellulose (250 mM KCI) 3B Sephadex G-150
1.2
" The 0-40% ammonium sulfate fraction obtained from 120g of wheat germ contains 1000 mg of protein,
tion of fraction 2B (3 ml containing 6 mg of protein) is concentrated to 1 ml by dialysis overnight against buffer B-100 containing 50% glycerol and is applied to a 100-ml Sephadex G-150 column (1.5 × 60 cm) equilibrated in buffer B-100. The column is developed with the same buffer, and fractions of 2 ml are collected at a flow rate of 20 ml/hr, The activity elutes from the column at the void volume. The active fractions are either stored or dialyzed overnight against buffer B- 100 containing 50% glycerol and stored at - 15°. A summary of the purification of fractions A and B is given in Table lI. F r a c t i o n a t i o n of the 4 0 - 6 0 % A m m o n i u m Sulfate F r a c t i o n (Fig. 1)
Step I. Separation of Fractions C, D, and E by Chromatography on DEAL-Cellulose. The 40-60% ammonium sulfate fraction of the 120 m M KC1 supernatant (25 ml containing 1800 mg of protein) is diluted to a protein concentration of about 35 mg/ml by the addition of buffer B-50 and is applied to a 60-ml DEAL-cellulose column (1. I × 60 cm) equilibrated in buffer B-50. The column is washed with the same buffer and is then developed successively with buffer B containing 120 and 250 m M KCI. Fractions of 6 ml are collected at a flow rate of 50 ml/hr. The first 8-10 fractions containing protein that is not retained by the column in 50 m M KC1 are pooled, concentrated about 3-fold by ultrafiltration with an Ami-
200
[14|
INITIATION OF PROTEIN SYNTHESIS
con PM-10 filter, and stored in small aliquots. This fraction is designated I C. All the fractions containing protein eluted by 120 mM KCI (generally 5-8 fractions) are pooled and designated fraction 1D. It is important that all the fractions containing significant amounts of protein be pooled, since there is some indication that two factors may be eluted by 120 mM KC1, one of which elutes behind the major peak of protein. The first 3 or 4 fractions containing protein eluted by 250 m M KCI are pooled, concentrated about 3-fold by ultrafiltration with an Amicon PM-10 filter, and stored in small aliquots. This fraction is designated IE. The separation of fractions C, D, and E by chromatography on DEAE-cellulose is summarized in Table III. Step 2D. Chromatography of Fraction 1D on Phosphocellulose. Two preparations of fraction ID obtained from two DEAE-cellulose columns are pooled (70 ml containing 440 mg of protein) and applied to a 40-ml phosphocellulose column (1.5 x 25 cm) equilibrated in buffer B-120. The column is washed with the same buffer and is then developed successively with buffer B containing 250 and 500 mM KCI. Fractions of 4 ml are collected at a flow rate of about 25 ml/hr. The activity is eluted by 500 mM KC1 and most of the activity is recovered in one or two fractions. This fraction is designated 2D. Step 3D. Chromatography of Fraction 2D on Sephadex G-150. Three milliliters of fraction 2D are concentrated by dialysis overnight against B-100 containing 50% glycerol. This material (1.0 ml containing 6 mg of protein) is applied to a 100-ml Sephadex G-150 column (1.5 X 60 cm) equilibrated in buffer B-100 and the column is developed with the same buffer. Fractions of 2 ml are collected at a flow rate of 20 ml/hr. The activity elutes from the column at 1.2-1.3 times the void volume. The purification of the factor in fraction D, which forms a ternary complex with Met-tRNAf and GTP, is summarized in Table IV.
TABLE III FRACTIONATION OF THE 40--60% AMMONIUM SULFATE FRACTION ON DEAE-CELLULOSE"
Fraction
KCI (raM)
Total protein (rag)
Total units ( × 10-'~)
Specific activity (units/rag)
1C ID IE
50 120 250
332 220 206
75 6 33
226 27 160
" The 40-60% ammonium sulfate fraction (1.8 g of protein) obtained from !20 g of wheat germ is applied to the DEAE-cellulose column.
[14]
201
W H E A T GERM I N I T I A T I O N FACTORS
E
E
¢q
O
r--
'5
~X
g~ Z ©
E E
>7-
I
¢q Z ©
Y:
r~
¢q
,~
E
C)
r~
Z
O
r~
r~
~2
202
I N I T I A T I O N OF P R O T E I N S Y N T H E S I S
[14]
Assay Methods
Polymerization Assay. The standard polymerization assay system contains in a total volume of 0.1 or 0.15 ml: 24 mM H E P E S - K O H , pH 7.6, 2.4 mM dithiothreitol, 0. ! mM spermine, 3.5 mM Mg(OAc).~, 85-90 mM KCI, 3-9% glycerol, 18 ~ [~4C]leucine (approximately 160 cpm/pmol), 50/All of the other 19 amino acids, 1 mM ATP (neutralized with KOH), 0.2 mM GTP, 7.8 mM creadne phosphate, 3 p+g of creatine phosphokinase, 0.75 A.,~0 unit of wheat germ tRNA, 1.2 A.~0 units of yeast polysomal RNA, 1.8-2.5A260 units of washed wheat germ ribosomes, 300 p,g of 20 mM KC1 postribosomal supernatant and fractions derived from the 120 mM KCI postribosomal supernatant as indicated below. The reaction mixture is incubated for 30 rain at 25°. The reaction is terminated by the addition of 3 mi of 5% trichloroacetic acid and heated for 10 min at 90°. The heated mixture is passed through a Millipore filter (type HA, 0.45/zm) and the filter is washed with two 3-ml portions of 1% trichloroacetic acid and one 3-ml portion of water. The filter is dried by heating at 100° for 7 min, placed in a vial containing 15 ml of scintillation fluid (5 g of PPO per liter of toluene), and the radioactivity is measured in a liquid scintillation counter. A unit is defined as ! pmol of [~4C]leucine incorporated into polypeptide under the conditions described. To assay for the various activities, the standard reaction mixture is supplemented as follows: Fraction A activity: 450/zg of the 40-60% ammonium sulfate fraction and 10/zg of 2B Fraction B activity: 450/~g of the 40-60% ammonium sulfate fraction and 14/zg of 2A Fraction C activity: 14 ~g of 2A, l0/zg of2B, 6 p,g of 2D, and 250/zg of 1E Fraction D activity: 14 ~g of 2A, 10 ~g of 2B, 160/zg of 1C, and 250/zg of 1E Fraction E activity: 14/.~gof2A, 10p+gof2B, 160~g of IC, and 6p+g of 2D Incorporation of [l+C]leucine into polypeptide is linear up to 1.5 hr. The amount of polymerization obtained is reduced about 2-fold when spermine is omitted and is highly dependent upon the concentrations of KCI, Mg '~+, and glycerol. The KC1 concentration is carefully maintained between 87 and 93 mM, taking into consideration the KCI contributed by all samples added to the assay. The optimum Mg 2+ concentration is between 3 and 4 mM. High concentrations of glycerol are inhibitory, and the total glycerol concentration in the assay is therefore kept below 9%. The amount of 20 mM KC1 supernatant used in the assay to supply elongation factors and
[14]
203
W H E A T GERM I N I T I A T I O N FACTORS
aminoacyl-tRNA synthetases is based on the amount required to support near maximal poly(U)-directed polyphenylalanine synthesis. Ternary Complex Assay. The standard ternary complex (elF-2-MettRNAf GTP) assay system contains in a total volume of 0.1 ml: 24 mM HEPES-KOH, pH 7.6, 100 mM KC1, 2.4 mM dithiothreitol, 0.02 mM GTP, 10-15 pmol of yeast [3'~S]Met-tRNAf9 (500-5000 cpm/pmol), and fraction D. The reaction mixture is incubated for 5 min at 25°. The reaction is terminated by the addition of 2 ml of cold buffer containing 20 mM Tris. HCI, pH 7.7, and 100 mM KC1, and is passed through a Millipore filter (type HA, 0.45/zm). The filter is washed with two 2-mi portions of the same buffer and dried, and the radioactivity is measured as described above. A unit is defined as 1 pmol of [35S]Met-tRNAf retained on the filter under the conditions described. Properties of the Purified Factors The dependence of polypeptide synthesis on the various fractions obtained from the 120 mM KC1 supernatant is shown in Table V. As shown in TABLE V DEPENDENCE OF POLYPEPTIDE SYNTHESIS ON VARIOUS FRACTIONS OBTAINED FROM THE 120 m M KC1 POSTRIBOSOMAL SUPERNATANT ° In the presence of untreated 20 m M KCI supernatant
In the presence of heparin-Sepharose-treated 20 m M KCI supernatant
Fraction omitted
['4C]Leucine incorporated (pmol)
Decrease (fold)
['4C]Leucine incorporated (pmol)
Decrease (fold)
None A B C D E
17 9 4 6 9 2
-1.9 4.3 2.8 1.9 8.5
14 2 1 5 4 2
-7.0 14.0 2.8 3.5 7.0
" T h e complete standard reaction mixture is s u p p l e m e n t e d with preparations of the variotis fractions as follows: 14 pg of 2A, 10/xg of 2B, 160/xg of IC, 7/xg of 2D, and 250/zg of 1E, and 0.32 mg of 20 m M KCI s u p e r n a t a n t or 0.43 mg of h e p a r i n - S e p h a r o s e - t r e a t e d 20 m M KCI supernatant as indicated. The values given above are corrected for the a m o u n t of polymerization obtained w h e n all the fractions are omitted (2 pmol with the untreated supernatant and 1 pmol with the h e p a r i n - S e p h a r o s e - t r e a t e d supernatant). W h e n all five fractions are a s s a y e d without a n y s u p e r n a t a n t being present, 5 pmol of polymerization are observed.
204
INITIATION
OF PROTEIN
SYNTHESIS
[15]
the table, a greater dependence on the fractions is obtained when heparin-Sepharose-treated 20 mM KC1 supernatant is used as a source of elongation factors and aminoacyi-tRNA synthetases rather than the untreated supernatant. Factor in Fraction A. Electrophoresis of fraction 3A (Sephadex G-150 fraction) on SDS polyacrylamide gels shows that this fraction contains two major species of protein, one with a molecular weight of approximately 80,000 and one greater than 100,000. Correlation of the activity with the intensity of the protein bands present in consecutive fractions obtained from Sephadex G-150 indicates that the active component is the 80,000 molecular weight species, strongly suggesting that this factor is comparable to elF-4B from rabbit reticulocytes. Benne et al. J5 have shown that elF-4B from rabbit reticulocytes is in the 0-40% ammonium sulfate fraction and is eluted from DEAE-cellulose and phosphocellulose at comparable salt concentrations. Factor in Fraction B. Glycerol density gradient centrifugation of fraction 2B indicates that the active component in this fraction has an s value of approximately 17 S. SDS polyacrylamide gel electrophoresis of fraction 3B shows 9-11 bands ranging in molecular weight from about 35,000 to 160,000. These data indicate that the active component in this fraction corresponds to elF-3 from mammalian cells. Factor in Fraction D. As previously reported, fraction D contains elF-2, the factor that forms a ternary complex with Met-tRNAe and GTP. Factors in Fractions C and E. These fractions have not been purified sufficiently for identification of the active components. Each of these fractions may contain more than one initiation factor and probably contains some elongation factors and aminoacyl-tRNA synthetases. ~ R. B e n n e , M. L u e d i , a n d J. W. B. H e r s h e y , J. Biol. Chem. 252, 5798 (1977).
[15] P l e i o t r o p i c E f f e c t s o f I n i t i a t i o n F a c t o r I F - 1 By H. O. VOORMA, R. BENNE, N. NAAKTGEBOREN,
and G. A. J. M. VAN DER HOESTAD Initiation factor IF-1 has a considerable effect on several steps in the initiation of protein synthesis in Escherichia coll. Since the molecular weight is only 9000 it is reasonable to assume that these different effects are caused by one basic action of IF-I which influences the reaction rates. The different effects of IF-1 are as follows: 1. Increase of the binding of IF-2 and fMet-tRNA to the 30 S subunit 2. Increase of the rate of recycling of IF-2
METHODS IN ENZYMOLOGY, VOL. LX
Copyright © 1979 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4