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[20] Purification of eukaryotic initiation factor 1 (EIF1) from Artemia saliva embryos
[20]
PURIFICATIONOF EUKARYOTIC INITIATION FACTOR 1
197
tion with deacylated-tRNA:~'~; moreover, aminoacyl-tRNA is deacylated at a finite rate during the assay. Thus, the deacylated-tRNA reaction is always likely to complicate the in vitro assay. For these reasons, it is important to understand the nature and the limitations of the reassoeiation assay. ~ R. D. Mosteller, W. J. Culp, and B. Hardesty, J. Biol. Chem. 243, 6343 (1968).
[20]
Purification
of Eukaryotic
from Artemia
Initiation
Factor
1 (EIF1)
salina Embryos
B y MICHAEL ZASLOFF and SEVERO OCHOA
Eukaryotic initiation factor 1 ( E I F 1 ) is a protein factor isolated from high speed supernatants of encysted embryos of the brine shrimp A rtemia salina which, like the bacterial polypeptide chain initiation factor IF2, promotes the AUG-dependent binding of fMet-tRNAr or the p o l y ( U ) dependent binding of N-acetyl-Phe-tRNA to the small ribosomal subunit. 1-~ The presence of a similar factor in rat liver supernatants has been reported by others? ,~ The rat liver and A . salina supernatant factors and ribosomal subunits are fully interchangeable suggesting that the two factors are probably identical. 2 An analogous factor, active with A . salina 40 S ribosomal subunits was demonstrated in the postribosomal supernatant of mouse fibroblastsY Moreover, partially purified preparations of the mammalian initiation factor M1 from 0.5 M KCI ribosomal washes of rabbit reticulocytes ~ or calf brain s ribosomes can replace the A . salina factor for fMet-tRNAf binding to A . salina 40 S ribosomal subunitsY ,4 A . salina EIF1 can also replace M1 in p o l y ( U ) translation by reticulocyte ribosomes at low Mg '-'÷ concentrationY M. Zasloff and S. Ochoa, Proc. Nat. Acad. Sci. U.S. 68, 3059 (1971). "~M. Zasloff and S. Ochoa, Proc. Nat. Acad. Sci. U.S. 69, 1796 (1972). 3R. P. McCroskey, M. Zasloff, and S. Ochoa, Proc. Nat. Acad. Sci. U.S. 69, 2457 (1972). "M. Zasloff and S. Ochoa, J. Mol. Biol. 73, 65 (1973). D. P. Leader and I. B. Wool, Biochim. Biophys. Acta 9.62, 360 (1972). ~E. Gasior and K. Moldave, J. Mol. Biol. 66, 391 (1972). "D. A. Shafritz, D. G. Laycock, R. G. Crystal, and W. F. Anderson, Proc. Nat. Acad. Sci. U.S. 68, 2246 (1971). 8S. S. Kerwar, C. Spears, and H. Weissbach, Biochem. Biophys. Res. Commun. 41, 78 (1970). 9 D. J. Picciano, P. M. Prichard, W. C. Merrick, D. A. Shafritz, H. Graf, R. G. Crystal, and W. F. Anderson, J. Biol. Chem. 248, 204 (1973).
198
INITIATION FACTORS IN PROTEIN SYNTHESIS
[20]
Assay The routine assay of EIF1 is based on its requirement for either the AUG-directed binding of fMet-tRNAf or the poly(U)-directed binding of N-acetyl-Phe-tRNA to Artemia salina 40 S ribosomal subunits/-4
Assay 1 Principle. The EIFl-dependent binding of f[I'C]Met-tRNA~ to A. salina 40 S ribosomal subunits, with AUG as messenger, is measured by the Millipore filter assay of Nirenberg and Leder. TM Reagents. The reaction mixtures, in a total volume of 0.06 ml, have the following composition: Tris.HC1 buffer, 90 mM, pH 7.5 (25 °) KC1, 150 mM Magnesium acetate, 4 mM Dithiothreitol (DTT), 1.5 mM AUG (Miles Laboratories), 0.05 A2~0 units A. salina 40 S ribosomal subunits, 0.3 ,4_~60units; EIF1, 0-2 units f[~'C]Met-tRNA (E. coli W, 400 cpm/pmole),l 20 pmoles
Procedure. The above components are mixed at 0 ° in the order listed. The reaction is started by the addition of f['~C]Met-tRNA. After incubation for 20 minutes at 0 ° the reaction is stopped by the addition of 3 ml of an ice-cold buffer containing 50 mM Tris.HC1, pH 7.75 (25°), 60 mM KC1, and 5 mM magnesium acetate, and the mixture is immediately filtered through a Millipore membrane (HAWP, 25 mm, 0.45 ~ pore size). The filters are washed with 12 ml of the diluting buffer and dried under an infrared lamp. The retained radioactivity is measured in 10 ml of Omnifluor (New England Nuclear Corp.) in a Packard Tri-Carb liquid scintillation spectrometer. Assay 2 Principle. The EIFl-dependent binding of N-acetyl[l'C]Phe-tRNA to A. salina 40 S ribosomal subunits with poly(U) as messenger is measured by the Millipore filter assay. Reagents. The reaction mixtures, in a total of 0.06 ml, have the following composition: Tris-HC1 buffer, pH 7.5 (25°), 90 mM KCI, 150 mM Magnesium acetate, 7 mM ~°M. W. Nirenberg and P. Leder, Science 145, 1399 (1964).
[20]
PURIFICATION OF EUKARYOTIC INITIATION FACTOR 1
199
DTT, 1.5 mM Poly(U) (Miles Laboratories), 1.0 A2G0 unit A. salina 40 S ribosomal subunits, 0.3 A.oG0unit; EIF1, 0-2 units N-acetyl[l'C]Phe-tRNA (E. coli W, 680 cpm/pmole), '~ 12 pmoles Procedure. The above components, except N-acetyl-Phe-tRNA, are mixed at 0 ° in the order listed. The reaction mixture is brought to 22 °, and the reaction is started by the addition of N-acetyl-Phe-tRNA. After incubation for 20 minutes the reaction is stopped by the addition of 3 ml of an ice-cold buffer containing 50 mM Tris.HC1, pH 7.75 (25°), 60 mM KC1, and 10 mM magnesium acetate, then filtered through Millipore membranes, washed with 12 ml of the diluting buffer, and finally dried and counted as described for Assay 1. One unit of EIFI activity is defined as the amount of protein that promotes the net binding (blanks without factor being subtracted) of 1 pmole of either fMet-tRNAr or N-acetyl-Phe-tRNA under the conditions of the respective assay. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. ~ EIF1 assays at approximately the same specific activity in both reaction systems, and either assay can be used reliably in the purification of EIF1. Under the conditions described, the rate of aminoacyl-tRNA binding is linear up to about 2.5 units/assay. Preparation of A. salina Ribosomal Subunits Materials A. salina cysts ("Brine Shrimp Eggs," a product of Longlife Aquar-
ium Products, Harrison, New Jersey, purchased from the Aquarium Stock Co., Inc., 31 Warren St., New York, N.Y.) Buffer A: Tris.HC1, 35 mM, pH 7.4 (25°); KC1, 70 mM; magnesium acetate, 9 mM; EDTA, 0.1 raM; 2-mercaptoethanol, 10 raM; sucrose, 250 mM Buffer B: Tris.HC1, 50 mM, pH 7.8 (25°); KC1, 700 mM; magnesium acetate, 11 mM; 2-mercaptoethanol, 20 mM Buffer C: Tris.HC1, 50 mM, pH 7.8 (25°); KC1, 200 mM; magnesium acetate, 10 mM; EDTA, 0.1 mM; 2-mercaptoethanol, 20 mM; sucrose, 250 mM Procedure. All operations are conducted at 0-4 ° . Thirty grams (dry weight) of A. salina cysts are suspended in 100 ml of ice-cold 1%
1~O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
200
INITIATION FACTORS IN PROTEIN SYNTHESIS
[9.0]
NaC10, stirred for 5 minutes, and then diluted into 1 liter of ice-cold distilled water. On standing, the cysts rapidly settle, leaving in the supernatant damaged organisms and other debris. The supernatant is decanted and the sediment is washed 10 times, each time in about 400 ml of water to ensure complete removal of the hypochlorite. The pretreatment of the cysts with hypochlorite sterilizes the preparation and, in addition, facilitates the grinding step by removing the granular part of the shelllz without damaging the embryo. The cysts are washed finally in 200 ml of buffer A, sedimented, resuspended in about 50 ml of buffer A, and then disrupted by grinding in a mortar. The thick paste is filtered through a single layer of cheese cloth which has been prewetted with buffer A, and the cloth filter is wrung to ensure complete drainage of the crude extract. One hundred milliliters of crude extract is generally obtained. The suspension is centrifuged for 15 minutes at 17,000 rpm in the Sorvall SS34 rotor to remove debris, nuclei, and mitochondria. The supernatant is carefully pipetted from above the pellet, filtered through a pad of glass wool, and then centrifuged for 30 minutes at 30,000 rpm in the Spinco No. 30 rotor to free the preparation from membranous debris and glycogen, which would otherwise sediment with the ribosomes. The upper threefourths of the supernatant is collected and centrifuged for 130 minutes at 50,000 rpm in the Spinco Ti 60 rotor. The upper two-thirds of the high speed supernatant is collected and stored at - 2 0 °. This supernatant can be used as the starting material for the purification, on a limited scale, of EIF1 and the chain elongation factors EF1 and EF2.1 The ribosomal pellet, which consists exclusively of the 80 S species, ~ is covered by a thin, dear, orange-tinted layer of membranous material, which is readily separated from the preparation by storing the pellets in ice for about 2 hours, after which time this material loosens from the surface of the dense, clear, and colorless ribosomal pellet. The pellets are rinsed gently with buffer B and then suspended in this buffer to a concentration of 400 A280 units/ml. Generally, about 1600 A~60 units of 80 S ribosomes are obtained. One milliliter of this suspension is immediately layered on each of three 50-ml 15 to 30% (w/v) sucrose gradients in buffer B and centrifuged for 14 hours at 24,000 rpm in the Spinco SW 25.2 rotor. Under these conditions, the 80 S ribosomes dissociate completely into 40 S and 60 S subunits. 1 Fractions comprising the faster 40% of the 60 S peak and the slower sedimenting 70% of the 40 S peak are pooled and immediately diluted with one volume of a buffer containing 30 mM magnesium acetate and 20 mM 2-mercaptoethanol. The subunits are concentrated to a clear Hi. E. Morris and B. A. Afzelius, 1. Ultrastruc. Res. 20, 244 (1967).
[20]
PURIFICATION OF EUKARYOTIC INITIATION FACTOR 1
201
pellet by centrifugation for 10 hours at 40,000 rpm in the Spinco Ti 60 rotor. It is important to note that, if the centrifugation is carried out at higher speeds, the resulting pellets yield, rather than a faintly opalescent solution, a milky, turbid suspension of virtually inactive ribosomes. The supernatants are discarded and the clear, colorless pellets are rinsed gently with buffer C. The 40 S and 60 S subunits are suspended in buffer C to a concentration of 200 and 400 A._,60units/ml, respectively, and stored at - 2 0 ° after adding one volume of glycerol. The subunits retain full activity in either the EIFl-dependent assays or in poly(U) translation for several months. As assayed by poly(U) translation, the 40 S subunits show less than 2-5%, and the 60 S subunits less than 5 %, contamination with the other species. When an equimolar mixture of subunits is suspended in buffer A they reassociate extensively to 80 S ribosomes. Purification Procedure Materials A. salina cysts
Phosphocellulose (Whatman P-11 ), 7.4 meq/g Sephadex G-200 (Pharmacia), 40--120/~ Carboxymethylcellulose (Whatman CM-52), 1.0 meq/g Hydroxyapatite (BioRad HTP) Buffer A: 60 mM KC1; 30 mM Tris.HC1, pH 7.75 (25°); 9 mM magnesium acetate; 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer B: 100 mM KC1; 50 mM Tris.HC1, pH 7.75 (25°); 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer C: 150 mM KC1; 50 mM Tris.HCl, pH 7.75 (25°); 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer D: 280 mM KC1; 50 mM Tris.HC1, pH 7.75 (25°); 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer E: 200 mM KCI; 50 mM Tris.HCl, pH 7.2 (4°); 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer F: 10 mM KCI; 30 mM Tris.HC1, pH 7.2 (4°); 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer G: I00 mM KCI; 30 mM Tris.HCl, pH 7.5 (25°); 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer H: 100 mM potassium phosphate, pH 7.15; 0.1 mM EDTA; 1 mM DTT; 10% glycerol (w/v) Buffer I: 170 mM potassium phosphate, pH 7.15; 0.1 mM EDTA; 1 mM DTT; 10% glycerol (w/v)
202
INITIATION FACTORS IN PROTEIN SYNTHESIS
[20]
Buffers are prepared by dilution of stock salt solutions which have been filtered through Millipore membranes (0.45 /~m pore size). Step 1. Preparation of Homogenate. All operations are conducted at 00-4 ° . Three kilograms of brine shrimp eggs were separately processed in 1-kg batches through the ammonium sulfate fractionation step. Commercial preparations of brine shrimp cysts generally contain large quantities of sea sand and other dense debris. Before the cysts can be disintegrated in a French press, the sand must be removed. This is readily accomplished by suspending 1 kg of cysts in 6 liters of saturated NaC1. The cysts, of lower density than the salt solution, rise slowly to the surface and are decanted, leaving behind the dense debris which has settled. This procedure is repeated until all sand has been removed. The suspension is then passed through a sieve (No. 80 U.S. Standard Series), and the cake is resuspended in about 4 liters of glass distilled water. The suspension is stirred gently and the cysts are allowed to settle, at which time the supernatant is poured off and a second portion of water is added. The washing is repeated until the preparation is free of salt. The cysts are then resuspended in about 1 liter of buffer A, allowed to settle, and finally resuspended in a total volume of 3200 ml of this buffer. The preparation is then disrupted by one passage through a French press at 6000-9000 psi. The chocolate-brown homogenate is centrifuged in the Sorvall GSA rotor at 10,000 rpm for 90 minutes. The supernatant, a milky-orange in color, is decanted and filtered through a layer of glass wool. Step 2. Acid Fractionation. To the crude homogenate (2400 ml) 1.0 N acetic acid is added dropwise with stirring to bring the pH of the suspension to 5.3. The preparation is stirred for about 5 minutes at which time it is cleared of precipitated material by centrifugation in the Sorvall GSA rotor at 10,000 rpm for 1 hour. The supernatant is decanted from the heavy orange precipitate and filtered through a layer of glass wool. The pH of the preparation is then adjusted to 6.8 by dropwise addition of 1.0 N KOH. As an alternative to acid fractionation, the crude homogenate can be centrifuged at 100,000 g for about 3 hours. The factor activity partitions primarily in the postribosomal supernatant and is of a specific activity comparable to that of the preparation obtained by the acid step. Step 3. Ammonium Sulfate Fractionation. Finely powdered ammonium sulfate (668 g) is added to the solution from the previous step (2050 ml) to 55% saturation at 0 °. The suspension is stirred for at least 1 hour and then allowed to settle overnight. The precipitate is collected by centrifugation in the Sorvall GSA rotor, at 10,000 rpm for 1 hour, and discarded. The supernatant (about 2000 ml) is brought to 78% saturation by addition of 354 g of ammonium sulfate and stirred for about 2 hours;
[20]
PURIFICATION OF EUKARYOTIC INITIATION FACTOR 1
203
the precipitate is harvested by low speed centrifugation. The precipitate is resuspended in about 100 ml of buffer B to a concentration of about 80 mg/ml. The brown turbid solution contains large amounts of high molecular weight glycogen, which is removed by centrifugation in the 60 Ti Spinco rotor at 50,000 rpm for 90 minutes. The clear, deep-brown supernatant is carefully pipetted off the colorless, viscous layer at the bottom of the tube and stored in ice. The three l-kg batches are generally worked up over three 2-day intervals, and the undialyzed ammonium sulfate cuts are stored in ice during the interim periods. After the third batch of cysts has been processed through the ammonium sulfate step, the ammonium sulfate fractions are pooled and dialyzed against several changes in buffer B (up to a volume of 15 liters), until the conductivity of the solution equals that of the buffer. Step 4. Phosphocellulose Chromatography. Phosphocellulose was washed with 25% NaC1, until the washes were colorless, and then with distilled water to remove the salt, packed into a column (8 X 24 cm), and equilibrated in buffer B. The solution from the preceding step (490 ml, 46 mg protein/ml) is applied to the column followed by washing with one column volume of buffer B, and then with buffer C, until the A..,,0 of the effluent falls to about 0.03 unit above that of the buffer. This removes large amounts of inactive protein. The activity is eluted from the column with buffer D, fractions of 10 ml being collected. The factor emerges slightly behind the main protein peak. Fractions of highest specific activity are pooled (110 ml) and the protein is concentrated by precipitation with ammonium sulfate (66 g) to 90% saturation. The suspension is stirred overnight and the precipitate is dissolved in about 20 ml of buffer E. This solution, containing about 18 mg/ml of protein, is dialyzed against 10 volumes of buffer E. The preparation can be frozen at this point and stored at - 2 0 ° for at least 1 month without appreciable loss of activity. Step 5. Gel Filtration on Sephadex G-200. The solution from the preceding step is applied to a column (2.5 × 90 cm) of Sephadex G-200. This column is developed with buffer E at a flow rate of 0.2 ml/min. Fractions of 3 ml are collected. The peak of factor activity elutes at approximately 1.5 x the void volume of the column. Fractions of highest specific activity are pooled and concentrated by ultrafiltration in an Amicon cell, fitted with a PM 30 membrane, to a volume of about 15 ml. This solution contains 16 mg/ml of protein. Although the increase in specific activity achieved on G-200 is rarely greater than 2-fold, this step is essential in that it effectively separates EIF1 from substantial amounts of EF1 and other high molecular weight proteins which will otherwise contaminate EIF1 in the subsequent steps.
204
INITIATION FACTORS IN PROTEIN SYNTHESIS
[20]
Step 6. Carboxymethyl Cellulose Chromatography. The protein solution from the previous step is dialyzed with several changes against a total of 2 liters of buffer F, equilibration being carefully ascertained by conductivity measurements. The solution is then applied to a column (2.5 x 40 cm) of carboxymethyl cellulose previously equilibrated in buffer F. The column is washed free of nonadsorbing protein with buffer F until the A28o of the effluent is negligible. The column is then developed with a linear salt gradient generated from 800 ml of buffer F and 800 ml of buffer G. The flow rate is maintained at 0.6 ml/minute and 10-ml fractions are collected. The factor elutes as a symmetrical peak centered approximately half way through the gradient. In dilute solutions near neutral p H the factor appears to be more stable at KCI concentrations in the neighborhood of 100 mM. Thus, 1 ml of a buffer containing 1 M KCI, 50 m M Tris.HCl, p H 7.2 ( 4 ° ) , 0.2 m M E D T A , and 5 m M fl-mercaptoethanol is included in each collecting tube. Fractions of highest specific activity are pooled and the protein concentrated by ultrafiltration to a volume of 12.5 ml at a concentration of 0.97 mg/ml. Step 7. Hydroxyapatite Chromatography. An aliquot of the solution of the preceding step (5 m g ) , dialyzed against 2 liters of buffer H overnight, is applied to a column (0.6 × 17 cm) of hydroxyapatite previously equilibrated in buffer H. The column is washed with this buffer until the A~80 of the effluent is negligible. It is then developed with buffer I at a flow rate of 0.15 ml per minute. Elution is continued until all the protein PURIFICATIONOF SUPERNATANTCHAIN INITIATIONFACTOR (EIF1) FROM Artemia salina EMBRYOS Step 1. Crude extract s 2. pH 5.3 supernatant 3. (NH4)2S04 fractionation 4. Phosphocellulose chromatography 5. Gel filtration on Sephadex G-200 6. CM-cellulose chromatography 7. Hydroxyapatite chromatographya
Volume (ml) 7500 5730 490 21
Protein (mg)
Unitsb Specific (X 10-4) activityc
220,000 103,510 22,700 372
-104 60 26
15
242
18
13
12
2.4
0.5
--
Yield (%)
10 26 703
-100 58 25
730
17
4.3
3,610
4
1.6
31,300
1.5
a From 3 kg (dry weight) of A. salina cysts. b One unit = 1 pmole of fMet-tRNA (Escherichia coli) bound under standard assay conditions (20 minutes at 0°). Units per milligram of protein. Based on 12 mg of step 6 factor.
[20]
PURIFICATION OF EUKARYOTIC INITIATION FACTOR
1
205
has emerged, 1.2-ml fractions being collected. Faster moving contaminants elute at the buffer front while EIF1 activity elutes as a broad peak of low protein concentration after approximately three column volumes (15 ml) have emerged. The fractions of highest specific activity are pooled and concentrated, from 40 ml to 5 ml by ultrafiltration and then further to 1 ml by dialysis against a concentrated solution of Carbowax 6000 (Union Carbide) in buffer B. The protein solution is made 50% (w/v) in glycerol and stored at - 2 0 °. A summary of the purification procedure is given in the table. Properties As a 50% glycerol solution in buffer B, stored at - 2 0 °, step 7 EIF1 shows no loss of activity over a period of 6 months, even at protein concentrations as low as 50 tzg/ml. Stock solutions of step 7 factor are generally diluted immediately prior to assay to a working concentration of about 15 ~g/ml (500 units/ml) in a solution of the composition of buffer B and containing 0.25 mg/ml bovine serum albumin (Armour). The procedure described yields a preparation of EIF1 purified over 3000-fold over the crude extract. On SDS gel electrophoresis step 7 factor gives rise to a single major band representing about 70-80% of the staining material and several minor components. The preparation is free of EF1 and EF2. On SDS gel electrophoresis the major band displays the mobility of a polypeptide of molecular weight about 74,000, while under nondenaturing conditions it elutes from Sephadex G-200 at a volume consistent with that of a globular protein of molecular weight about 145,000. We thus infer that A . salina EIF1 is composed of two subunits of equivalent mass.' EIF1 requires a free 40 S ribosomal subunit for expression of its activity. 1-' Thus, it will catalyze the template directed binding of fMet-tRNAf and N-acetyl-Phe-tRNA solely to the free 40 S ribosomal subunit, but not to either the 60 S or 80 S species. The N-acylaminoacyl-tRNA is bound to the 40 S subunit on a site defined as the initiation site since the subsequent addition of the 60 S subunit and puromycin results in the direct conversion of the bound N-acylaminoacyl-tRNA to the dipeptide analog, N-acylaminoacylpuromycin? Both the binding reaction and the synthesis of the puromycin derivatives occur in the absence of GTP or other ribonucleoside triphosphates. 3 EIF1 will also promote the template-directed binding of Met-tRNAr and Phe-tRNA to the 40 S ribosomal subunit; however, the bound aminoacyl-tRNA's are found to be less reactive with puromycin than their corresponding N-blocked derivatives? Equivalent results have been obtained with tRNA species from either E. coli or A. salina. :~ Under the conditions of the standard binding assays, EIF1 has been
206
INITIATION FACTORS IN PROTEIN SYNTHESIS
[21]
shown to function catalytically.4 The reaction is inhibited by low concentrations of edeine and aurintricarboxylic acid, inhibitors of polypeptide chain initiation in both eukaryotic and prokaryotic systems.1,2 EIF1 is sensitive to SH binding reagents such as N-ethylmaleimide and p-chloromercuribenzoate. 1,~ The prokaryotic and eukaryotic initiation factors IF2 and EIF1 are not interchangeable2 even though they catalyze identical reactions in their respective systems.
[21] A s s a y a n d I s o l a t i o n of a 40 S R i b o s o m a l S u b u n i t " M e t - t R N A ~ et B i n d i n g F a c t o r from Rabbit Reticulocytes
By J. M. CIMADEVILLAand B. HARDESTY A protein factor from rabbit reticulocytes promotes codon-directed binding of various forms of tRNA Phe and tRNA~ e* to 40 S ribosomal subunits. 1 The factor with poly(U) or ApUpG is active with either the N-acetylaminoacyl-tRNA, aminoacyl-tRNA or the deacylated forms of these species of tRNA. It appears to have little or no activity for MettRNA~ e* or other species of tRNA tested. Factor-dependent binding of tRNA to the 40 S ribosomal subunit is severely inhibited by the 60 S subunit. This factor-dependent binding also is inhibited by aurintricarboxylic acid and edeine, both of which inhibit peptide initiation on eukaryotic ribosomes. GTP or other ribonucleoside triphosphates have no effect on binding under any conditions tested. Met-tRNA~ °t bound to 40 S subunits with the binding factor is sensitive to hydrolysis by Met-tRNA hydrolase considered elsewhere in this volume. The tRNA binding factor prepared as described below is virtually free of EF-1, EF-2, Phe-tRNA synthetase, and Met-tRNA synthetase. It appears to be a basic protein of molecular weight about 85,000 that is sensitive to inactivation by sulfhydryl reactive reagents and heat. The factor considered here appears to be similar or identical to tRNA binding factors from brine shrimp, 2 rat liver, ~,4 ascites cells, 5 wheat, ~ and 1 j. M. Cimadevilla and B. Hardesty, unpublished observations. ~M. Zasloff and S. Ochoa, Proc. Nat. Acad. Sci. U.S. 68, 3059 (1971). ~E. Gasior and K. Moldave, J. Mol. Biol. 66, 391 (1972). ' D . Leader and I. Wool, Biochim. Biophys. Acta 9.,62, 360 (1972). 5D. Leader, H. Klein-Brehaar, I. Wool, and A. Fox, Biochem. Biophys. Res. Commun. 46, 215 (1972). 6A. Marcus, .L Biol. Chem. 245, 962 (1970).
PURIFICATIONOF EUKARYOTIC INITIATION FACTOR 1
197
tion with deacylated-tRNA:~'~; moreover, aminoacyl-tRNA is deacylated at a finite rate during the assay. Thus, the deacylated-tRNA reaction is always likely to complicate the in vitro assay. For these reasons, it is important to understand the nature and the limitations of the reassoeiation assay. ~ R. D. Mosteller, W. J. Culp, and B. Hardesty, J. Biol. Chem. 243, 6343 (1968).
[20]
Purification
of Eukaryotic
from Artemia
Initiation
Factor
1 (EIF1)
salina Embryos
B y MICHAEL ZASLOFF and SEVERO OCHOA
Eukaryotic initiation factor 1 ( E I F 1 ) is a protein factor isolated from high speed supernatants of encysted embryos of the brine shrimp A rtemia salina which, like the bacterial polypeptide chain initiation factor IF2, promotes the AUG-dependent binding of fMet-tRNAr or the p o l y ( U ) dependent binding of N-acetyl-Phe-tRNA to the small ribosomal subunit. 1-~ The presence of a similar factor in rat liver supernatants has been reported by others? ,~ The rat liver and A . salina supernatant factors and ribosomal subunits are fully interchangeable suggesting that the two factors are probably identical. 2 An analogous factor, active with A . salina 40 S ribosomal subunits was demonstrated in the postribosomal supernatant of mouse fibroblastsY Moreover, partially purified preparations of the mammalian initiation factor M1 from 0.5 M KCI ribosomal washes of rabbit reticulocytes ~ or calf brain s ribosomes can replace the A . salina factor for fMet-tRNAf binding to A . salina 40 S ribosomal subunitsY ,4 A . salina EIF1 can also replace M1 in p o l y ( U ) translation by reticulocyte ribosomes at low Mg '-'÷ concentrationY M. Zasloff and S. Ochoa, Proc. Nat. Acad. Sci. U.S. 68, 3059 (1971). "~M. Zasloff and S. Ochoa, Proc. Nat. Acad. Sci. U.S. 69, 1796 (1972). 3R. P. McCroskey, M. Zasloff, and S. Ochoa, Proc. Nat. Acad. Sci. U.S. 69, 2457 (1972). "M. Zasloff and S. Ochoa, J. Mol. Biol. 73, 65 (1973). D. P. Leader and I. B. Wool, Biochim. Biophys. Acta 9.62, 360 (1972). ~E. Gasior and K. Moldave, J. Mol. Biol. 66, 391 (1972). "D. A. Shafritz, D. G. Laycock, R. G. Crystal, and W. F. Anderson, Proc. Nat. Acad. Sci. U.S. 68, 2246 (1971). 8S. S. Kerwar, C. Spears, and H. Weissbach, Biochem. Biophys. Res. Commun. 41, 78 (1970). 9 D. J. Picciano, P. M. Prichard, W. C. Merrick, D. A. Shafritz, H. Graf, R. G. Crystal, and W. F. Anderson, J. Biol. Chem. 248, 204 (1973).
198
INITIATION FACTORS IN PROTEIN SYNTHESIS
[20]
Assay The routine assay of EIF1 is based on its requirement for either the AUG-directed binding of fMet-tRNAf or the poly(U)-directed binding of N-acetyl-Phe-tRNA to Artemia salina 40 S ribosomal subunits/-4
Assay 1 Principle. The EIFl-dependent binding of f[I'C]Met-tRNA~ to A. salina 40 S ribosomal subunits, with AUG as messenger, is measured by the Millipore filter assay of Nirenberg and Leder. TM Reagents. The reaction mixtures, in a total volume of 0.06 ml, have the following composition: Tris.HC1 buffer, 90 mM, pH 7.5 (25 °) KC1, 150 mM Magnesium acetate, 4 mM Dithiothreitol (DTT), 1.5 mM AUG (Miles Laboratories), 0.05 A2~0 units A. salina 40 S ribosomal subunits, 0.3 ,4_~60units; EIF1, 0-2 units f[~'C]Met-tRNA (E. coli W, 400 cpm/pmole),l 20 pmoles
Procedure. The above components are mixed at 0 ° in the order listed. The reaction is started by the addition of f['~C]Met-tRNA. After incubation for 20 minutes at 0 ° the reaction is stopped by the addition of 3 ml of an ice-cold buffer containing 50 mM Tris.HC1, pH 7.75 (25°), 60 mM KC1, and 5 mM magnesium acetate, and the mixture is immediately filtered through a Millipore membrane (HAWP, 25 mm, 0.45 ~ pore size). The filters are washed with 12 ml of the diluting buffer and dried under an infrared lamp. The retained radioactivity is measured in 10 ml of Omnifluor (New England Nuclear Corp.) in a Packard Tri-Carb liquid scintillation spectrometer. Assay 2 Principle. The EIFl-dependent binding of N-acetyl[l'C]Phe-tRNA to A. salina 40 S ribosomal subunits with poly(U) as messenger is measured by the Millipore filter assay. Reagents. The reaction mixtures, in a total of 0.06 ml, have the following composition: Tris-HC1 buffer, pH 7.5 (25°), 90 mM KCI, 150 mM Magnesium acetate, 7 mM ~°M. W. Nirenberg and P. Leder, Science 145, 1399 (1964).
[20]
PURIFICATION OF EUKARYOTIC INITIATION FACTOR 1
199
DTT, 1.5 mM Poly(U) (Miles Laboratories), 1.0 A2G0 unit A. salina 40 S ribosomal subunits, 0.3 A.oG0unit; EIF1, 0-2 units N-acetyl[l'C]Phe-tRNA (E. coli W, 680 cpm/pmole), '~ 12 pmoles Procedure. The above components, except N-acetyl-Phe-tRNA, are mixed at 0 ° in the order listed. The reaction mixture is brought to 22 °, and the reaction is started by the addition of N-acetyl-Phe-tRNA. After incubation for 20 minutes the reaction is stopped by the addition of 3 ml of an ice-cold buffer containing 50 mM Tris.HC1, pH 7.75 (25°), 60 mM KC1, and 10 mM magnesium acetate, then filtered through Millipore membranes, washed with 12 ml of the diluting buffer, and finally dried and counted as described for Assay 1. One unit of EIFI activity is defined as the amount of protein that promotes the net binding (blanks without factor being subtracted) of 1 pmole of either fMet-tRNAr or N-acetyl-Phe-tRNA under the conditions of the respective assay. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al. ~ EIF1 assays at approximately the same specific activity in both reaction systems, and either assay can be used reliably in the purification of EIF1. Under the conditions described, the rate of aminoacyl-tRNA binding is linear up to about 2.5 units/assay. Preparation of A. salina Ribosomal Subunits Materials A. salina cysts ("Brine Shrimp Eggs," a product of Longlife Aquar-
ium Products, Harrison, New Jersey, purchased from the Aquarium Stock Co., Inc., 31 Warren St., New York, N.Y.) Buffer A: Tris.HC1, 35 mM, pH 7.4 (25°); KC1, 70 mM; magnesium acetate, 9 mM; EDTA, 0.1 raM; 2-mercaptoethanol, 10 raM; sucrose, 250 mM Buffer B: Tris.HC1, 50 mM, pH 7.8 (25°); KC1, 700 mM; magnesium acetate, 11 mM; 2-mercaptoethanol, 20 mM Buffer C: Tris.HC1, 50 mM, pH 7.8 (25°); KC1, 200 mM; magnesium acetate, 10 mM; EDTA, 0.1 mM; 2-mercaptoethanol, 20 mM; sucrose, 250 mM Procedure. All operations are conducted at 0-4 ° . Thirty grams (dry weight) of A. salina cysts are suspended in 100 ml of ice-cold 1%
1~O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
200
INITIATION FACTORS IN PROTEIN SYNTHESIS
[9.0]
NaC10, stirred for 5 minutes, and then diluted into 1 liter of ice-cold distilled water. On standing, the cysts rapidly settle, leaving in the supernatant damaged organisms and other debris. The supernatant is decanted and the sediment is washed 10 times, each time in about 400 ml of water to ensure complete removal of the hypochlorite. The pretreatment of the cysts with hypochlorite sterilizes the preparation and, in addition, facilitates the grinding step by removing the granular part of the shelllz without damaging the embryo. The cysts are washed finally in 200 ml of buffer A, sedimented, resuspended in about 50 ml of buffer A, and then disrupted by grinding in a mortar. The thick paste is filtered through a single layer of cheese cloth which has been prewetted with buffer A, and the cloth filter is wrung to ensure complete drainage of the crude extract. One hundred milliliters of crude extract is generally obtained. The suspension is centrifuged for 15 minutes at 17,000 rpm in the Sorvall SS34 rotor to remove debris, nuclei, and mitochondria. The supernatant is carefully pipetted from above the pellet, filtered through a pad of glass wool, and then centrifuged for 30 minutes at 30,000 rpm in the Spinco No. 30 rotor to free the preparation from membranous debris and glycogen, which would otherwise sediment with the ribosomes. The upper threefourths of the supernatant is collected and centrifuged for 130 minutes at 50,000 rpm in the Spinco Ti 60 rotor. The upper two-thirds of the high speed supernatant is collected and stored at - 2 0 °. This supernatant can be used as the starting material for the purification, on a limited scale, of EIF1 and the chain elongation factors EF1 and EF2.1 The ribosomal pellet, which consists exclusively of the 80 S species, ~ is covered by a thin, dear, orange-tinted layer of membranous material, which is readily separated from the preparation by storing the pellets in ice for about 2 hours, after which time this material loosens from the surface of the dense, clear, and colorless ribosomal pellet. The pellets are rinsed gently with buffer B and then suspended in this buffer to a concentration of 400 A280 units/ml. Generally, about 1600 A~60 units of 80 S ribosomes are obtained. One milliliter of this suspension is immediately layered on each of three 50-ml 15 to 30% (w/v) sucrose gradients in buffer B and centrifuged for 14 hours at 24,000 rpm in the Spinco SW 25.2 rotor. Under these conditions, the 80 S ribosomes dissociate completely into 40 S and 60 S subunits. 1 Fractions comprising the faster 40% of the 60 S peak and the slower sedimenting 70% of the 40 S peak are pooled and immediately diluted with one volume of a buffer containing 30 mM magnesium acetate and 20 mM 2-mercaptoethanol. The subunits are concentrated to a clear Hi. E. Morris and B. A. Afzelius, 1. Ultrastruc. Res. 20, 244 (1967).
[20]
PURIFICATION OF EUKARYOTIC INITIATION FACTOR 1
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pellet by centrifugation for 10 hours at 40,000 rpm in the Spinco Ti 60 rotor. It is important to note that, if the centrifugation is carried out at higher speeds, the resulting pellets yield, rather than a faintly opalescent solution, a milky, turbid suspension of virtually inactive ribosomes. The supernatants are discarded and the clear, colorless pellets are rinsed gently with buffer C. The 40 S and 60 S subunits are suspended in buffer C to a concentration of 200 and 400 A._,60units/ml, respectively, and stored at - 2 0 ° after adding one volume of glycerol. The subunits retain full activity in either the EIFl-dependent assays or in poly(U) translation for several months. As assayed by poly(U) translation, the 40 S subunits show less than 2-5%, and the 60 S subunits less than 5 %, contamination with the other species. When an equimolar mixture of subunits is suspended in buffer A they reassociate extensively to 80 S ribosomes. Purification Procedure Materials A. salina cysts
Phosphocellulose (Whatman P-11 ), 7.4 meq/g Sephadex G-200 (Pharmacia), 40--120/~ Carboxymethylcellulose (Whatman CM-52), 1.0 meq/g Hydroxyapatite (BioRad HTP) Buffer A: 60 mM KC1; 30 mM Tris.HC1, pH 7.75 (25°); 9 mM magnesium acetate; 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer B: 100 mM KC1; 50 mM Tris.HC1, pH 7.75 (25°); 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer C: 150 mM KC1; 50 mM Tris.HCl, pH 7.75 (25°); 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer D: 280 mM KC1; 50 mM Tris.HC1, pH 7.75 (25°); 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer E: 200 mM KCI; 50 mM Tris.HCl, pH 7.2 (4°); 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer F: 10 mM KCI; 30 mM Tris.HC1, pH 7.2 (4°); 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer G: I00 mM KCI; 30 mM Tris.HCl, pH 7.5 (25°); 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 10% glycerol (w/v) Buffer H: 100 mM potassium phosphate, pH 7.15; 0.1 mM EDTA; 1 mM DTT; 10% glycerol (w/v) Buffer I: 170 mM potassium phosphate, pH 7.15; 0.1 mM EDTA; 1 mM DTT; 10% glycerol (w/v)
202
INITIATION FACTORS IN PROTEIN SYNTHESIS
[20]
Buffers are prepared by dilution of stock salt solutions which have been filtered through Millipore membranes (0.45 /~m pore size). Step 1. Preparation of Homogenate. All operations are conducted at 00-4 ° . Three kilograms of brine shrimp eggs were separately processed in 1-kg batches through the ammonium sulfate fractionation step. Commercial preparations of brine shrimp cysts generally contain large quantities of sea sand and other dense debris. Before the cysts can be disintegrated in a French press, the sand must be removed. This is readily accomplished by suspending 1 kg of cysts in 6 liters of saturated NaC1. The cysts, of lower density than the salt solution, rise slowly to the surface and are decanted, leaving behind the dense debris which has settled. This procedure is repeated until all sand has been removed. The suspension is then passed through a sieve (No. 80 U.S. Standard Series), and the cake is resuspended in about 4 liters of glass distilled water. The suspension is stirred gently and the cysts are allowed to settle, at which time the supernatant is poured off and a second portion of water is added. The washing is repeated until the preparation is free of salt. The cysts are then resuspended in about 1 liter of buffer A, allowed to settle, and finally resuspended in a total volume of 3200 ml of this buffer. The preparation is then disrupted by one passage through a French press at 6000-9000 psi. The chocolate-brown homogenate is centrifuged in the Sorvall GSA rotor at 10,000 rpm for 90 minutes. The supernatant, a milky-orange in color, is decanted and filtered through a layer of glass wool. Step 2. Acid Fractionation. To the crude homogenate (2400 ml) 1.0 N acetic acid is added dropwise with stirring to bring the pH of the suspension to 5.3. The preparation is stirred for about 5 minutes at which time it is cleared of precipitated material by centrifugation in the Sorvall GSA rotor at 10,000 rpm for 1 hour. The supernatant is decanted from the heavy orange precipitate and filtered through a layer of glass wool. The pH of the preparation is then adjusted to 6.8 by dropwise addition of 1.0 N KOH. As an alternative to acid fractionation, the crude homogenate can be centrifuged at 100,000 g for about 3 hours. The factor activity partitions primarily in the postribosomal supernatant and is of a specific activity comparable to that of the preparation obtained by the acid step. Step 3. Ammonium Sulfate Fractionation. Finely powdered ammonium sulfate (668 g) is added to the solution from the previous step (2050 ml) to 55% saturation at 0 °. The suspension is stirred for at least 1 hour and then allowed to settle overnight. The precipitate is collected by centrifugation in the Sorvall GSA rotor, at 10,000 rpm for 1 hour, and discarded. The supernatant (about 2000 ml) is brought to 78% saturation by addition of 354 g of ammonium sulfate and stirred for about 2 hours;
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the precipitate is harvested by low speed centrifugation. The precipitate is resuspended in about 100 ml of buffer B to a concentration of about 80 mg/ml. The brown turbid solution contains large amounts of high molecular weight glycogen, which is removed by centrifugation in the 60 Ti Spinco rotor at 50,000 rpm for 90 minutes. The clear, deep-brown supernatant is carefully pipetted off the colorless, viscous layer at the bottom of the tube and stored in ice. The three l-kg batches are generally worked up over three 2-day intervals, and the undialyzed ammonium sulfate cuts are stored in ice during the interim periods. After the third batch of cysts has been processed through the ammonium sulfate step, the ammonium sulfate fractions are pooled and dialyzed against several changes in buffer B (up to a volume of 15 liters), until the conductivity of the solution equals that of the buffer. Step 4. Phosphocellulose Chromatography. Phosphocellulose was washed with 25% NaC1, until the washes were colorless, and then with distilled water to remove the salt, packed into a column (8 X 24 cm), and equilibrated in buffer B. The solution from the preceding step (490 ml, 46 mg protein/ml) is applied to the column followed by washing with one column volume of buffer B, and then with buffer C, until the A..,,0 of the effluent falls to about 0.03 unit above that of the buffer. This removes large amounts of inactive protein. The activity is eluted from the column with buffer D, fractions of 10 ml being collected. The factor emerges slightly behind the main protein peak. Fractions of highest specific activity are pooled (110 ml) and the protein is concentrated by precipitation with ammonium sulfate (66 g) to 90% saturation. The suspension is stirred overnight and the precipitate is dissolved in about 20 ml of buffer E. This solution, containing about 18 mg/ml of protein, is dialyzed against 10 volumes of buffer E. The preparation can be frozen at this point and stored at - 2 0 ° for at least 1 month without appreciable loss of activity. Step 5. Gel Filtration on Sephadex G-200. The solution from the preceding step is applied to a column (2.5 × 90 cm) of Sephadex G-200. This column is developed with buffer E at a flow rate of 0.2 ml/min. Fractions of 3 ml are collected. The peak of factor activity elutes at approximately 1.5 x the void volume of the column. Fractions of highest specific activity are pooled and concentrated by ultrafiltration in an Amicon cell, fitted with a PM 30 membrane, to a volume of about 15 ml. This solution contains 16 mg/ml of protein. Although the increase in specific activity achieved on G-200 is rarely greater than 2-fold, this step is essential in that it effectively separates EIF1 from substantial amounts of EF1 and other high molecular weight proteins which will otherwise contaminate EIF1 in the subsequent steps.
204
INITIATION FACTORS IN PROTEIN SYNTHESIS
[20]
Step 6. Carboxymethyl Cellulose Chromatography. The protein solution from the previous step is dialyzed with several changes against a total of 2 liters of buffer F, equilibration being carefully ascertained by conductivity measurements. The solution is then applied to a column (2.5 x 40 cm) of carboxymethyl cellulose previously equilibrated in buffer F. The column is washed free of nonadsorbing protein with buffer F until the A28o of the effluent is negligible. The column is then developed with a linear salt gradient generated from 800 ml of buffer F and 800 ml of buffer G. The flow rate is maintained at 0.6 ml/minute and 10-ml fractions are collected. The factor elutes as a symmetrical peak centered approximately half way through the gradient. In dilute solutions near neutral p H the factor appears to be more stable at KCI concentrations in the neighborhood of 100 mM. Thus, 1 ml of a buffer containing 1 M KCI, 50 m M Tris.HCl, p H 7.2 ( 4 ° ) , 0.2 m M E D T A , and 5 m M fl-mercaptoethanol is included in each collecting tube. Fractions of highest specific activity are pooled and the protein concentrated by ultrafiltration to a volume of 12.5 ml at a concentration of 0.97 mg/ml. Step 7. Hydroxyapatite Chromatography. An aliquot of the solution of the preceding step (5 m g ) , dialyzed against 2 liters of buffer H overnight, is applied to a column (0.6 × 17 cm) of hydroxyapatite previously equilibrated in buffer H. The column is washed with this buffer until the A~80 of the effluent is negligible. It is then developed with buffer I at a flow rate of 0.15 ml per minute. Elution is continued until all the protein PURIFICATIONOF SUPERNATANTCHAIN INITIATIONFACTOR (EIF1) FROM Artemia salina EMBRYOS Step 1. Crude extract s 2. pH 5.3 supernatant 3. (NH4)2S04 fractionation 4. Phosphocellulose chromatography 5. Gel filtration on Sephadex G-200 6. CM-cellulose chromatography 7. Hydroxyapatite chromatographya
Volume (ml) 7500 5730 490 21
Protein (mg)
Unitsb Specific (X 10-4) activityc
220,000 103,510 22,700 372
-104 60 26
15
242
18
13
12
2.4
0.5
--
Yield (%)
10 26 703
-100 58 25
730
17
4.3
3,610
4
1.6
31,300
1.5
a From 3 kg (dry weight) of A. salina cysts. b One unit = 1 pmole of fMet-tRNA (Escherichia coli) bound under standard assay conditions (20 minutes at 0°). Units per milligram of protein. Based on 12 mg of step 6 factor.
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1
205
has emerged, 1.2-ml fractions being collected. Faster moving contaminants elute at the buffer front while EIF1 activity elutes as a broad peak of low protein concentration after approximately three column volumes (15 ml) have emerged. The fractions of highest specific activity are pooled and concentrated, from 40 ml to 5 ml by ultrafiltration and then further to 1 ml by dialysis against a concentrated solution of Carbowax 6000 (Union Carbide) in buffer B. The protein solution is made 50% (w/v) in glycerol and stored at - 2 0 °. A summary of the purification procedure is given in the table. Properties As a 50% glycerol solution in buffer B, stored at - 2 0 °, step 7 EIF1 shows no loss of activity over a period of 6 months, even at protein concentrations as low as 50 tzg/ml. Stock solutions of step 7 factor are generally diluted immediately prior to assay to a working concentration of about 15 ~g/ml (500 units/ml) in a solution of the composition of buffer B and containing 0.25 mg/ml bovine serum albumin (Armour). The procedure described yields a preparation of EIF1 purified over 3000-fold over the crude extract. On SDS gel electrophoresis step 7 factor gives rise to a single major band representing about 70-80% of the staining material and several minor components. The preparation is free of EF1 and EF2. On SDS gel electrophoresis the major band displays the mobility of a polypeptide of molecular weight about 74,000, while under nondenaturing conditions it elutes from Sephadex G-200 at a volume consistent with that of a globular protein of molecular weight about 145,000. We thus infer that A . salina EIF1 is composed of two subunits of equivalent mass.' EIF1 requires a free 40 S ribosomal subunit for expression of its activity. 1-' Thus, it will catalyze the template directed binding of fMet-tRNAf and N-acetyl-Phe-tRNA solely to the free 40 S ribosomal subunit, but not to either the 60 S or 80 S species. The N-acylaminoacyl-tRNA is bound to the 40 S subunit on a site defined as the initiation site since the subsequent addition of the 60 S subunit and puromycin results in the direct conversion of the bound N-acylaminoacyl-tRNA to the dipeptide analog, N-acylaminoacylpuromycin? Both the binding reaction and the synthesis of the puromycin derivatives occur in the absence of GTP or other ribonucleoside triphosphates. 3 EIF1 will also promote the template-directed binding of Met-tRNAr and Phe-tRNA to the 40 S ribosomal subunit; however, the bound aminoacyl-tRNA's are found to be less reactive with puromycin than their corresponding N-blocked derivatives? Equivalent results have been obtained with tRNA species from either E. coli or A. salina. :~ Under the conditions of the standard binding assays, EIF1 has been
206
INITIATION FACTORS IN PROTEIN SYNTHESIS
[21]
shown to function catalytically.4 The reaction is inhibited by low concentrations of edeine and aurintricarboxylic acid, inhibitors of polypeptide chain initiation in both eukaryotic and prokaryotic systems.1,2 EIF1 is sensitive to SH binding reagents such as N-ethylmaleimide and p-chloromercuribenzoate. 1,~ The prokaryotic and eukaryotic initiation factors IF2 and EIF1 are not interchangeable2 even though they catalyze identical reactions in their respective systems.
[21] A s s a y a n d I s o l a t i o n of a 40 S R i b o s o m a l S u b u n i t " M e t - t R N A ~ et B i n d i n g F a c t o r from Rabbit Reticulocytes
By J. M. CIMADEVILLAand B. HARDESTY A protein factor from rabbit reticulocytes promotes codon-directed binding of various forms of tRNA Phe and tRNA~ e* to 40 S ribosomal subunits. 1 The factor with poly(U) or ApUpG is active with either the N-acetylaminoacyl-tRNA, aminoacyl-tRNA or the deacylated forms of these species of tRNA. It appears to have little or no activity for MettRNA~ e* or other species of tRNA tested. Factor-dependent binding of tRNA to the 40 S ribosomal subunit is severely inhibited by the 60 S subunit. This factor-dependent binding also is inhibited by aurintricarboxylic acid and edeine, both of which inhibit peptide initiation on eukaryotic ribosomes. GTP or other ribonucleoside triphosphates have no effect on binding under any conditions tested. Met-tRNA~ °t bound to 40 S subunits with the binding factor is sensitive to hydrolysis by Met-tRNA hydrolase considered elsewhere in this volume. The tRNA binding factor prepared as described below is virtually free of EF-1, EF-2, Phe-tRNA synthetase, and Met-tRNA synthetase. It appears to be a basic protein of molecular weight about 85,000 that is sensitive to inactivation by sulfhydryl reactive reagents and heat. The factor considered here appears to be similar or identical to tRNA binding factors from brine shrimp, 2 rat liver, ~,4 ascites cells, 5 wheat, ~ and 1 j. M. Cimadevilla and B. Hardesty, unpublished observations. ~M. Zasloff and S. Ochoa, Proc. Nat. Acad. Sci. U.S. 68, 3059 (1971). ~E. Gasior and K. Moldave, J. Mol. Biol. 66, 391 (1972). ' D . Leader and I. Wool, Biochim. Biophys. Acta 9.,62, 360 (1972). 5D. Leader, H. Klein-Brehaar, I. Wool, and A. Fox, Biochem. Biophys. Res. Commun. 46, 215 (1972). 6A. Marcus, .L Biol. Chem. 245, 962 (1970).