ARCHIVES

OF BIOCHEMISTRY

Purification

JOHN

Department

AND

BIOPHYSICS

182,

626-638

(1977)

and Characterization of Protein Synthesis Initiation Factors IFI, IF2, and IF3 from Escherichia co/i

W. B. HERSHEY,’

of Biological

Chemistry,

JOAN YANOV, KATHLEEN JOHN L. FAKUNDINGZ School

of Medicine,

Received

University

December

of California,

JOHNSTON

Davis,

AND

California

95616

22, 1976

A simple procedure is described for the purification in high yields of protein synthesis initiation factors IFl, IF2, and IF3 from Escherichia coli strain MRE 600. IF2 was separated from IF1 and IF3 by ammonium sulfate fractionation and was purified by column chromatography on phosphocellulose and diethylaminoethyl (DEAE) Sephadex. IF1 and IF3 were separated by phosphocellulose column chromatography. IF1 was purified by molecular sieve chromatography, and IF3 by phosphocellulose column chromatography in urea buffer. Each factor was analyzed by sodium dodecyl sulfate or urea polyacrylamide gel electrophoresis and was greater than 98% pure. Only one form of IF1 and IF3 was found, with molecular weights of 8,500 and 22,500, respectively. Two forms of IF2 were isolated: IF2a with a molecular weight of 118,000 and IF2b with a molecular weight of 90,000. The amino acid composition of each factor was determined, and their stimulation in a variety of assays for initiation of protein synthesis is reported.

Initiation of protein synthesis in Escherichia coli is promoted by three protein factors. These are found associated with ribosomes but can be separated from them by centrifugation in buffer containing high salt. The initiation factors, called IFl, IF2 and IF3,3 have been studied extensively during the past 10 years and the broad features of their role in initiation of protein synthesis have been elucidated. Detailed descriptions of the factors are found in recent reviews (l-4). Current studies in this laboratory include the identification of ribosomal binding sites for factors by cross-linking techniques, the effects of chemical modification on factor activities, and the preparation and use of specific antibodies against these proteins. Highly purified and characterized factor proteins 1 Author to whom reprint requests should be sent. * Present address: Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77025. 3 Abbreviations used: IF, initiation factor; DEAE, diethylaminoethyl; DNase, deoxyribonuclease; RNase, ribonuclease; PPO, 2,5-diphenyloxazole; POPOP, 1,4-bis[2-(5-phenyloxazolyl)]benzene; SDS, sodium dodecyl sulfate.

are required in large amounts for all of these studies. In this report we describe a simple and rapid method for the purification of all three factors in high yield. The degree of purity is carefully monitored and factors greater than 98% pure are obtained. Each is characterized with respect to molecular weight, amino acid composition, and biological activity. All three factors stimulate protein synthesis or various partial reactions of the initiation process with phage R17 RNA as messenger RNA. EXPERIMENTAL

PROCEDURES

Materials. Biochemical compounds were obtained as follows: GTP from Calbiochem; dithiothreitol from Pierce Chemical Co.; glass beads, Superbrite 100, from 3M Company; DNase, RNase-free, from Worthington; phosphocellulose, P-11 (7.4 meqlg) from Whatman; DEAE-Sephadex, A-50 (3.5 meqlg) and Sephadex G-50, from Pharmacia. All other chemicals were reagent grade. Toluene scintillation fluid was prepared by mixing 4 g of 2,5-diphenyloxazole (PPO) and 0.05 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene (POPOP) per liter of toluene. A-U-G was prepared according to the method of Sundararajan and Thach (5). Formyl[14C]methionyl-tRNA was prepared from unfractionated E. coli B tRNA

626 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0003-9861

PROTEIN

SYNTHESIS

INITIATION

FACTORS

(Schwarz-Mann) and [‘*C]methionine (SchwarzMann, specific activity 265 Ci/mol) according to the method of Hershey and Thach (6). R17 RNA was prepared by phenol extraction of R17 phage essentially as described by Gesteland and Spahr (7). 70 S ribosomes were prepared and washed twice in high salt buffer as previously described (8). 30 S subunits were prepared by a modification (9) of a procedure described by No11 (10). Bz&rs. Buffer A contained: 10 mM potassium phosphate, pH 7.5; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 5% glycerol; and KCl. Buffer B contained: 10 mM Tris-HCl, pH 7.4; 0.1 mM Mg-acetate; 7 mM 2mercaptoethanol; 10% glycerol; 6 M urea, and NH&l. Various concentrations of KC1 or NH&l were used in these buffers and the millimolar concentration is shown in parentheses. For example, buffer A with 100 mM KC1 is denoted buffer A (100). Cell growth. E. coli strain MRE 600 was grown in a New Brunswick Model F-130 fermenter in 100-l&r batches in a buffered enriched medium containing, per liter: 11.6 g yeast extract (Difco), 42.0 g of KH,PO,, 14 g of KOH, and 10 g of glucose, final pH 7.0. Each batch was inoculated with 1 liter of stationary phase cells grown in the same medium overnight. Growth was carried out at 37°C with maximum stirring (780 rpm) and aeration (10 ft?min); foaming was suppressed by the addition of 50 ml of Antifoam A (Sigma). Growth was stopped in late log phase (A,,, = 6.5 in a Gilford 2400s spectrophotometer) by emptying the culture into 50 liters of crushed ice to bring the temperature to 0-4°C in less than 1 min. The chilled cells were harvested quickly by centrifugation in a Sharples continuous flow centrifuge, washed once by resuspension in 20 mM TrisHCl, pH 7.5, 10 mM Mg-acetate, and 0.5 mM EDTA, and pelleted in liter bottles at 8000 rpm in a Lourdes centrifuge. The cells, about 1 kg wet weight/lOOliter batch, were stored at -70” in 100-g amounts. Cell lysate. Frozen cells (500 g) were combined with 1250 g of glass beads and 500 ml of buffer (10 mM Tris-HCl, pH 7.4; 10 mM Mg-acetate) in a l-gal Waring Blendor and were mixed at high speed for a total of 15 min. The temperature was maintained at 0-5°C by the periodic addition of powdered dry ice. DNase (150 pg) was added and the beads were removed by centrifugation at 7000 rpm in a Sorvall GSA rotor. The beads were resuspended in 300 ml of buffer and centrifuged again. The supernatants from the two centrifugations were combined and centrifuged 20 min at 16,000 rpm in the Sorvall SS34 rotor. The clarified cell lysate (615 ml) was carefully removed from the pellet and held on ice for further fractionation as described under Results. Assays. Initiation factor-dependent binding of formylmethionyl-tRNA to 70 S ribosomes was used routinely to assay factor activity. Each assay mixture of 40 ~1 contained: 20 mM Tris-HCl, pH 7.4; 100

IFl,

IF2,

AND

IF3

FROM

E. coli

627

mM NH&l plus KCl; 5 to 7 mM Mg-acetate; 1 mM dithiothreitol; 1 mM GTP; 0.6Azso units of washed 70 S ribosomes; and 10 pg of unfractionated tRNA charged with 16 pmol [‘*C]methionine. For the IF1 activity assay, 20 pM A-U-G and 0.5 to 1.5 pg of IF2 were included. For IF2, 20 PM A-U-G and 0.1 to 0.2 pg of IF1 were added. For IF3, 15 pg of R17 RNA, LO-I.5 pg of IF2, and 0.15-0.2 pg of IF1 were added. The volume of factor assayed was usually 2 to 5 ~1. The reaction was started by the addition of a mixture containing the GTP, A-U-G, and formyl[14C]methionyl-tRNA to the other components, and was incubated 5 min at 30°C. The mixture was diluted with 1 ml of cold buffer (10 mM Tris-HCl, pH 7.5; 50 mM NH&l; 10 mM Mg-acetate) and filtered immediately through glass fiber filters Whatman GF/C, 2.4 cm). After washing and drying, the filters were placed in 5 ml of scintillation fluid and counted in a Beckman LS-200 scintillation counter at 90% efficiency. Phosphocellulose Column chromatography. (Whatman Pll) and DEAE-Sephadex (Pharmacia A-50) were treated as directed by the manufacturers and then equilibrated with buffer A (100). To the settled resins were added 2 vol of 0.1% bovine serum albumin in buffer A, the suspension was stirred 1 h, and the resin was filtered, washed thoroughly with buffer A (1000) to remove the bovine serum albumin, and equilibrated with buffer A (100). Phosphocellulose columns were packed tightly under 200 cm of hydrostatic pressure while DEAE-Sephadex A-50 columns were prepared at less than 40 cm hydrostatic pressure. Details of column size and elution conditions are given under Results. The salt concentrations in elutant fractions were determined with a conductivity meter (Radiometer) and absorbance at 280 nm was measured with a spectrophotometer (Gilford 2400). Amino acid analyses. The method used was essentially that of Moore and Stein (11). Acid hydrolysis of degassed protein samples (about 100 pg each) was carried out in 6 N HCl and 0.2% phenol at 110°C or 24, 48, and 72 h. Analyses of the hydrolysates were performed with a Beckman Model 121 automatic amino acid analyzer equipped with a Beckman Model 125 integrator. Valine and isoleucine values were obtained only from 72-h hydrolysates. Serine values were determined by use of a linear regression to zero time of the logarithms of the number of residues, while threonine was determined either by regression analysis (for IF3) or by 24-h hydrolysate values corrected for 5% loss (for IF1 and IF2a). Cysteine was analyzed as cysteic acid in performate-oxidized samples as described by Hirs (12), and also by the method of Ellman (13). Tryptophan was determined by analysis of samples hydrolyzed as described above but containing 4% mercaptoacetic acid (14). The numbers of moles of each

628

HERSHEY

amino acid recovered were normalized to that of glycine, averaged, and the mole percentages were calculated. The numbers of residues per molecule were calculated on the basis of molecular weights of 8,500, 118,000, and 22,500 for IFl, IF2a, and IFS, respectively, and an average residue weight of 112. RESULTS

Purification

AND DISCUSSION

of Initiation

Factors

Preparation of crude factors. One kilogram of E. coli MRE 600 cells was lysed as described under Experimental Procedures and was fractionated as follows. The lysate (1230 ml) was divided into 24 equal portions and layered over 25ml cushions containing 10 mu Tris-HCl, pH 7.4, 20 mM NH&l, 10 mu Mg-acetate, 7 mu 2-mercaptoethanol, and 15% glycerol in Beckman polycarbonate tubes. The solutions were centrifuged at 35,000 rpm for 10 h at 4°C in a Beckman type 35 rotor. The upper 80% of each supernatant was removed and saved as the S-100 fraction; the next 10% was discarded. The lowest 10% of the supernatant and the pellet were suspended in buffer and brought to 20 mu Tris-HCl, pH 7.4, 500 mM NH&l, 20 mM Mg-acetate, and 7 mM 2-mercaptoethanol in a final volume of 1000 ml. The solution was stirred 1 h and then layered over 30ml cushions of the same buffer containing 20% glycerol. The ribosome solutions were centrifuged 14 h as described above. The supernatant was decanted and saved as the 0.5 M salt wash. The pellets were resuspended in buffer and brought to 20 mu Tris-HCl, pH 7.4, 1000 mu NH&l, 40 mM Mg-acetate, and 7 mu 2-mercaptoethanol in a final volume of 1000 ml. This solution was centrifuged through a glycerol cushion as before to provide the 1 M salt wash and 20-30 g of pelleted ribosomes. As each wash was obtained, it was immediately subjected to ammonium sulfate fractionation (see below). Best results are obtained when delays are minimized; the elapsed time from cell lysis to the completion of the ammonium sulfate fractionation steps (below) should not be greater than 55 h. Separation of IF2 from IF1 and IF3 by ammonium sulfate fractionation. The 0.5

and 1.0 M salt washes were separately brought to 45% saturation by slow (1 h)

ET AL.

addition of a saturated ammonium sulfate solution at 4°C. After an additional hour of stirring, the precipitates were collected by centrifugation, and the supernatants were brought to 80% saturation by the slow addition of solid ammonium sulfate. The protein precipitate formed at 45% saturation from the 0.5 M salt wash contained most of the IF2; that from the 1.0 M salt wash contained less than 15% of the IF2 and was discarded since it contained proteins which are difficult to separate from IF2 by the procedures described below. The precipitate formed at 80% saturation from the 0.5 M salt wash contained most of the IF1 and IF3; that from the 1.0 M wash contained appreciable quantities of IF3 and was combined with the other. The crude initiation factors can be stored for several weeks as frozen (-70°C) ammonium sulfate pastes with little or no loss of activity. Purification of IF2. The precipitate formed at 45% saturated ammonium sulfate of the 0.5 M wash was dissolved in about 100 ml of buffer A (0) and dialyzed overnight against 2 x 4 liter of buffer A (100). The dialyzed solution was diluted to 200 ml and precipitated protein was removed by centrifugation. The soluble protein (780 mg) was adsorbed on a phosphocellulose column (1.5 x 95 cm) equilibrated with buffer A (loo), and was eluted with a 2-liter linear salt gradient in buffer A (100-500). One fraction (20 ml) was collected per hour. IF2 was detected by its stimulation of formylmethionyl-tRNA binding to 70 S ribosomes in the presence of A-U-G (see Experimental Procedures) and by SDS/polyacrylamide gel electrophoresis. The protein concentration and activity profiles were similar to those described previously (15). The IF2 activity was eluted from 320 to 400 mM KC1 and active fractions were combined to yield 84 mg of protein in 440 ml. The pooled fractions from the phosphocellulose column were diluted in a Tefloncoated beaker with 1 liter of buffer A (0) to bring the KC1 concentration to about 100 mM. The solution was adsorbed on a DEAE-Sephadex column (2.0 x 60 cm) equilibrated with buffer A (1001, and the protein was eluted with a 2-liter linear salt

PROTEIN

SYNTHESIS

INITIATION

FACTORS

gradient in buffer A (150-450). Fractions (20 ml) were collected at the rate of one per hour and were assayed for IF2 activity and analyzed by SDS/polyacrylamide gel electrophoresis. As shown in Fig. 1, the IF2 activity coincided with two distinct protein peaks. The first, which was eluted from 290 to 310 mM KCl, contained the larger molecular weight form, IF2a. The second was eluted from 320 to 350 mM KC1 and contained the smaller form, IF2b. IF2a is the predominant form (29 mg) and is usually about 90% pure at this stage of the purification. IF2b varies between 15 and 30% of total IF2, and is usually 70 to 80% pure. IF2a was obtained in a nearly homogeneous form by rechromatography on a phosphocellulose column (0.9 x 30 cm). The material above was diluted to 100 mM KC1 with buffer A (O), adsorbed to the column, and eluted with a 200-ml linear salt gradient in buffer A (150-500). Pure, active fractions were combined and stored frozen at - 70°C. Analysis of the protein by polyacrylamide gel electrophoresis in SDS or urea indicated that the IF2a was 98% pure (Fig. 4). Separation of IF1 from IF3. The protein precipitating between 45 and 80% ammonium sulfate saturation from both the 0.5 and 1.0 M salt washes was combined and dissolved in buffer A (0). The solution was dialyzed overnight against 2 x 4 liter of buffer A (loo), brought to 250 ml, and clar-

00

10

20I

x,I+J 40 50,-s, 60 Fraction Number

70

IFl,

IF2,

AND

IF3

FROM

E. coli

629

ified by centrifugation. The soluble protein (5.1 g) was applied to a phosphocellulose column (1.5 x 100 cm) equilibrated with buffer A (1001, and was eluted with a 2liter salt gradient in buffer A (100-1000). One fraction (20 ml) was collected per hour. Activity and absorbance profiles are shown in Fig. 2. IF1 was eluted in a sharp peak from 490 to 530 mM KC1 (fractions 4650; 99 mg of protein); IF3 was eluted in a broad peak from 680 to 810 mM KC1 (fractions 65-77; 46 mg of protein). Analysis of the pooled fractions by SDS/polyacrylamide gel electrophoresis showed that the IF1 was quite impure and that the IF3 was about 33% pure. Purification of IFl. In order to concentrate the impure IF1 fraction above, the solution was diluted with buffer A (0) to bring the KC1 concentration to 100 mM and was adsorbed onto a small phosphocellulose column (bed volume, 8 ml). The protein was eluted with buffer A (1000) and the fractions containing the bulk of the protein were pooled (volume, 7 ml). The concentrated IF1 was divided in half and each half was applied separately to a Sephadex G-50 column (1.5 x 100 cm) equilibrated with buffer A (200). The column

J0

FIG. 1. Chromatography of IF2 on DEAE-Sephadex. Procedures are given in the text. Aliquots of 5 ~1 were assayed as described under Experimental Procedures; no background blank was subtracted. Fractions 43-48 were pooled to yield IF2a; fractions 54-58 contained IF2b.

Fraction

Number

FIG. 2. Chromatography of IF1 and IF3 on phosphocellulose. Procedures are given in the text. Aliquota of 5 ~1 were assayed as described under Experimental Procedures. Minus factor blanks of 300 cpm were subtracted for both the IF1 and IF3 activity assays.

HERSHEY

630

was washed with the same buffer at 10 ml/ h and fractions (about 2 ml) were analyzed by measuring the absorbance at 280 mn and by SDWpolyacrylamide gel electrophoresis. IF1 was eluted later than the bulk of the protein (fractions 40-50; 9 mg of protein from both columns), and was either homogeneous or greater than 90% pure at this stage. If homogeneous IF1 was not obtained, the remaining impurities were removed by chromatography on a small phosphocellulose column (0.9 x 25 cm>. After this step IF1 is pure by the criterion of polyacrylamide gel electrophoresis in SDS or urea (Fig. 4). Purification of IF3. The IF3 fraction (33% pure) from the phosphocellulose column above was purified further by phosphocellulose column chromatography in 6 M urea essentially according to the procedure of Lee-Huang and Ochoa (18). IF3 (46 mg) was brought to 6 M urea with solid urea (SchwarzlMann, Ultrapure) and was dialyzed overnight against 2 x 1 liter of buffer B (30). The sample was adsorbed onto a column (0.9 x 30 cm) of phosphocellulose equilibrated with the same buffer and the protein was eluted with a 400~ml linear salt gradient in buffer B (100-500). Fractions of 4 ml were collected every 30 min and 2-~1 aliquots were analyzed directly for IF3 activity as described under Experimental Procedures. As shown in Fig. 3, the IF3 activity was eluted from 180 to 260 mM NH&l (fractions 28 to 45) as a broad, apparently double peak. When fractions 31 to 40 were diluted lo-fold and 2-~1

1 0.4 s 2 0.3 8 2 0.2 5 4” 0.1 0

0

7 600 % ” 5 &Q :z 4’ 2KJ5

0

102030405060608090

Fraction

Number

FIG. 3. Chromatography of IF3 on phosphocellulose in urea buffer. Procedures are given in the text. A minus factor blank of 400 cpm was subtracted.

ET AL.

aliquots were assayed, however, a single sharp peak with a maximum at fraction 34 was obtained (results not shown). Based on analysis by SDWpolyacrylamide gel electrophoresis, fractions 34-36 were combined to yield 3 mg of IF3 which was more than 95% pure (Fig. 4). Fractions 27-33 (6 mg; 70% pure) and 37-44 (8 mg; 25% pure) were pooled separately and each was chromatographed a second time on phosphocellulose in urea to remove contaminating proteins. IF3 activity was eluted at 190-230 mM NH&I in both cases, I SDS

UREA IF2a

IF1

IF3 IF1 I

I

\

J L FIG. 4. Densitometric tracings of stained polyacrylamide gels. The upper panel shows polyacrylamide gels run in SDS buffer as described by Bickle and Traut (16). The acrylamide concentrations in the gels were 5% (IF2a), 10% (IF31, and 15% (IFI). The lower panel shows scans of polyacrylamide gels containing 7.5% acrylamide and 0.2% bisacrylamide run in 8 M urea buffer at pH 4.4 as described by Leboy et al. (17), except that the stacking gel was omitted. For each gel, 4 to 8 pg of the most purified pooled fractions of each initiation factor were added; electrophoretic migration occurred from left to right. The gels were stained with Coomassie brilliant blue, destained and scanned in a Gilford 2400 spectrophotometer with a linear transport attachment. The IF3 sample was treated with iodoacetamide prior to analysis in the urea gel system; such treatment was necessary in order to prevent the formation of IF3 dimers during electrophoresis.

PROTEIN

SYNTHESIS

INITIATION

FACTORS

and the IF3 proteins obtained from the two sources were indistinguishable when analyzed for activity and by polyacrylamide gel electrophoresis in SDS or urea buffers. These results (not shown) suggest that only one form of IF3 was present in the apparently double activity peak shown in Fig. 3. Although the IF3 fractions in urea buffer can be assayed directly if small volumes (e.g. 2 ~1) are used, it is ultimately necessary to remove the urea by a procedure which does not cause a large loss of protein. Combined fractions in urea were diluted with 2 vol of water, and the protein was adsorbed on a small phosphocellulose column (1 to 2 ml bed volume). The column was washed with 20 ml of buffer A (100) to remove urea, and the protein was eluted with buffer A (800). This procedure serves not only to remove urea but also to concentrate the IF3 solution. Dilute solutions of TABLE SUMMARY Purification High salt 0.5 M 1.0 M

step

OF PURIFICATION Protein

(mg)

IFl,

IFZ,

AND

IF3

FROM

631

E. coli

IF1 and IF2 were concentrated in the same way. Yields and purity. The fractionation scheme for the three initiation factors is shown diagrammatically in Fig. 5. The amounts of protein and factor activities obtained from a typical preparation are shown in Table I. The extract from 1 kg of

+ IF20

FIG.

4

IF1

5. Fractionation

IF3

scheme

for

IFl,

IF2

and

IF3. I OF IFI,

IF2, Activity (units)

AND IF3” Specific activity (unitslmg)

Percentage yield

washes

IF2a Ammonium sulfate; Phosphocellulose DEAE-cellulose Phosphocellulose

7,020 2,214

-

-

-

O-45%

780 84 29 15.8

97 78 48 34

0.12 0.93 1.65 2.15

100 80 50 35

45-80%

5,125 99 9 4.7

61 36 27

0.62 4.0 5.7

100 59 44

Ammonium sulfate: 45-80% Phosphocellulose Phosphocellulose, urea

5,125 46 7.1

312 207 100

0.06 4.50 14.1

100 67 32

IF1 Ammonium sulfate: Phosphocellulose Sephadex G-50 Phosphocellulose IF3

n The values are derived from a typical preparation involving 1 kg of cells. Protein was determined by the method of Lowry et al. (20). Activity was determined by measuring formylmethionyl-tRNA binding to 70 S ribosomes in the presence of A-U-G (for IF1 and IF21 or R17 RNA (for IF31, as described under Experimental Procedures. One activity unit is defined as the ability to stimulate the ribosomal binding of one nanomole of formyl[14C]methionyl-tRNA. All determinations were made in the region of linear response of the factor. Since the absolute values for activity also depend on the quality of the ribosomes and other assay ingredients, and thus may vary from time to time, all values for a given factor were determined in the same experiment.

632

HERSHEY

cells routinely provides 4 to 7 mg of IFl, 15 to 25 mg of IF2a, and 5 to 10 mg of IF3. Based on activity measurements, these values represent 30 to 50% yields of each factor. Efficient recovery is possible because only two or three chromatographic procedures are required to obtain nearly homogeneous proteins. Other elements which contribute to the high yields are: (a) minimal delay between lysing the cells and beginning the first chromatographic columns; (b) the use of siliconized or Teflon-coated vessels whenever possible; (c) the treatment of chromatography resins with protein (e.g., bovine serum albumin or lysozyme) prior to use; (d) the avoidance of dialysis at later stages of the preparations (dialysis membranes bind all three factors); (e) the avoidance of dilute solutions of purified factors; and (f) the use of small phosphocellulose columns to concentrate factor preparations. Most loss of activity appears to be due to the loss of protein on vessel surfaces. This is readily apparent when radioactively labeled factors are employed. Most of the items above are addressed to this problem. The highly purified initiation factors were analyzed by polyacrylamide gel electrophoresis in buffers containing either SDS or urea. Densitometric tracings of the stained gels are shown in Fig. 4. The purity of each factor was calculated from the area under the peaks. IF1 and IF3 had no detectable contaminants; IF2a contained about 2% of IF2b. The proteins were also analyzed in a two-dimensional gel system which utilized a gel containing 4% acrylamide in urea buffer at pH 4.5 (17) in the first dimension, followed by electrophoresis in the second dimension in a slab containing 7.5% acrylamide in SDS buffer (19). The procedure did not reveal additional components (data not shown). Thus the relatively simple purification procedures described here provide essentially homogeneous IFl, IF2a, and IF3 in high yield. Comparison with other purification methods. Procedures have been described

previously for the purification to near homogeneity of the three bacterial initiation factors. Ochoa and co-workers (21-23) and

ET AL.

Dubnoff and Maitra (24) purified the factors from E. coli strain Q13, while Wahba and Miller (25) and Grunberg-Manago and co-workers (26) used E. coli strain MRE 600. It is of interest to compare these procedures with the method described in this report which also used E. coli strain MRE 600. Since large quantities of highly purified proteins are now required for much of the current research on initiation factors (e.g. chemical and physical studies, crosslinking, preparation of antibodies), we shall contrast the various methods with respect to simplicity, yield and purity of product. Specifically, we shall compare: (i) the number of chromatographic steps which follow the high salt extraction and ammonium sulfate fractionation steps which are similar in nearly all the methods; (ii) the yield of each factor, expressed here as milligrams of protein per kilogram of bacterial cells; and (iii) the purity of the preparations, usually determined by polyacrylamide gel electrophoresis in SDS buffer. The procedures developed and described relatively early by Ochoa and co-workers (21-23) allow the preparation of essentially homogeneous factors. Either three or four chromatographic steps are needed for each factor, and the yields are somewhat low, especially for IF2 (0.6 mg). The method of Dubnoff and Maitra (24) involves three steps per factor. However, yields are not reported and the IF2 preparation contained a number of contaminating proteins. Wahba and Miller (25) have described relatively simple methods for the purification of IF1 (four steps; 1.5 mg), IF2 (two steps; 3.4 mg of the large form), and IF3 (three steps; 3.9 mg); IF1 and IF3 were obtained in homogeneous form, but IF2a was only 82% pure. The procedures of Dondon et al. (26) differ from the others in that the ammonium sulfate step is omitted; only three steps per factor are needed to obtain nearly homogeneous IF1 (1.5 mg), IF2 (10 mg), and IF3 (3 mg). The procedures described in this report use many of the individual purification steps incorporated in the schemes previously described by others. Our method differs primarily in that phosphocellulose

PROTEIN

SYNTHESIS

INITIATION

FACTORS

rather than DEAE-cellulose is used in the first chromatographic step which follows ammonium sulfate fractionation. The use of phosphocellulose at this stage is advantageous because: (i) The resin has a high protein-binding capacity, which allows the use of smaller columns and results in less dilution of protein; (ii) the bulk of the protein fails to bind to phosphocellulose and a high degree of purification is obtained, and (iii) each factor elutes reproducibly in a single, rather narrow range of salt concentrations. Following phosphocellulose chromatography, only one additional step is required in order to obtain each factor in a state of purity exceeding 90%) while preparations greater than 98% pure are obtained in two additional steps. Furthermore, the average yields of homogeneous factors are two to five times greater than the best yields reported by others: for IFl, 5 mg; for IF2a, 20 mg; and for IF3, 8 mg. Our method involves two salt extractions of the ribosomes rather than one. However, the second extraction may be omitted without significantly affecting the purity or yields of IF1 and IF2, although the yield of IF3 is reduced by about 30%. Physicochemical

Properties

Multiple forms. Two forms of IF2 were obtained which differ in size: IF2a, molecular weight 118,000 (see below); and IFPb, molecular weight 90,000. It is likely that the same gene codes for both forms and that IF2a is converted into IF2b by limited proteolysis. The following observations support this view: (i) Treatment of the two forms of IF2 with cyanogen bromide or trypsin results in the generation of peptide fragments, most of which are identical (2); (ii) rabbit antibody prepared against IF2a cross-reacts with IF2b; (iii) both forms are phosphorylated by rabbit skeletal muscle protein kinase (27); (iv) when cell lysates are fractionated rapidly into ribosome and supernatant fractions and then analyzed by SDS/polyacrylamide gel electrophoresis, little or no IF2b is detected; and (v) relatively greater amounts of IF2b are obtained when the early fractionation procedures are not performed rapidly. It is therefore likely that IF2b is an artifact of

IFl,

IF!&

AND

IF3

FROM

E. coli

633

isolation and is not present in appreciable quantities in intact cells. No difference in initiation factor activity was detected. Beports of multiple IF2 proteins have been made previously by Kolakofsky et al. (28); Lelong et al. (29); Mazumder (30); Fakunding et al. (27); and Miller and Wahba (31). Only one form of IF1 or IF3 was isolated. Since two forms of IF3 have been reported in a number of laboratories (18, 31-33), special efforts were made to identify a second form. The various fractions from the ion-exchange columns were analyzed not only by using the assays described, but also by testing for their ability to bind radioactive poly(U,G) to Millipore filters. In addition, proteins with a molecular weight of 20,000-25,000 were sought by analyzing by SDS/polyacrylamide gel electrophoresis those fractions which might contain a second form. Our inability to detect multiple forms of IF3 is consistent with the observations of Schiff et al. (34) and Spremulli et al. (35). Molecular weights. Accurate molecular weights of the initiation factors are required in studies of their stoichiometric relationships and are useful in comparing one preparation of factor with another. To determine the molecular weights of the factors, polyacrylamide gel electrophoresis in SDS was employed essentially according to the method of Weber and Osborn (36). Plots of the logarithm of the molecular weight versus gel migration distance for protein standards and factors are shown in Fig. 6. The molecular weights determined in this way are listed in Table II. The molecular weight of IF1 was also determined by Sephadex G-50 gel filtration as described previously (37). A molecular weight value of 9,300 was obtained for the IF1 prepared from E. coli strain MBE 600 (data not shown), which is identical to that reported previously for IF1 isolated from E. coli strain DlO (37). The molecular weight of IF1 also can be calculated from the amino acid analysis data reported below. By assuming that the average residue molecular weight is 112 and that the molecule contains exactly two residues of histidine, phenylalanine, or tyrosine, values obtained are 8,100,8,200 and 8,500, respec-

634

HERSHEY

b 3 ::

20

% IO s 6 i t 4O

I 0.2 Relative

I I I 0.4 0.6 0.8 Migration Distance

1 I .o

FIG. 6. Molecular weight determinations by SDWpolyacrylamide gel electrophoresis. Determinations for IF1 and IF3 were made by using the phosphate buffer system described by Bickle and Traut (16) with disc gels containing either 15 (0) or 11% (0) acrylamide. For IF2a, either the same disc gel system containing 5% acrylamide (01 was employed, or a slab gel with 6.5% acrylamide (W) was run in Tris buffer according to the method of Laemmli (19). About 2 pg of each of the initiation factors and the following protein markers were used as indicated (molecular weight in parentheses): myosin heavy chain (220,000), P-galactosidase (130,000), phosphorylase A (94,000), bovine serum albumin (67,500), pyruvate kinase (57,000), ovalbumin (43,000), carbonic anhydrase (29,000), chymotrypsinogen A (255001, myosin (17,200), ribonuclease A (13,700), cytochrome c (12,400), and cyanogen bromide fragments of myoglobin (8,900 and 6,400). The positions of migration of the initiation factors are indicated by arrows. The molecular weight values are reported in Table 2.

tively. Alternatively, when the number of residues of each amino acid to the nearest integral value is multiplied by the actual residue molecular weight and the products are summed, a value of 8,700 is obtained. The values for IFl, which average about 8,700, are somewhat lower than those reported elsewhere: 9,400 by gel filtration, SDWpolyacrylamide gels, and sedimentation equilibrium (21); 9,207 by meniscus depletion ultracentrifugation (25); and 9,000 by SDWpolyacrylamide gels (38). The values of IF2a and IFBb, 118,000 (Fig. 6 and Table II) and 90,000 (determined as for IF2a; data not shown), respectively, are considerably higher than those

ET AL.

determined earlier by SDS/polyacrylamide gel electrophoresis: 91,000 and 82,000 (27) or 98,000 and 83,000 (31). The differences may have occurred because the earlier values were obtained by extrapolation from marker proteins of lower molecular weight than IF2a, while the determinations reported here included a number of marker proteins with molecular weights greater than IF2a. The molecular weight value for IF3 of about 22,500 (Fig. 6 and Table II) is in close agreement with those reported previously: 21,000 (381, 22,600 (251, or 21,500 and 23,500 for the two forms described by Lee-Huang and Ochoa (18). Amino acid composition. Amino acid analyses of initiation factor hydrolysates were performed as described under Experimental Procedures. The mole percentages and numbers of residues for each of the factors are reported in Table III. Except where cited, the values are the average of at least six determinations made on two different preparations of each factor. Values for ammonia were not reproducible and are not reported. The presence of trace amounts of glycerol (0.1 to 0.5%) during acid hydrolysis caused abnormally low values for glutamic acid, high values for threonine, and considerable variation in the values for those residues only. When pure glutamic acid was subjected to the hydrolysis conditions for 24 h in 0.8% glycerol, 63% was converted to a substance eluting in the position of threonine, 7% to a substance eluting like aspartic acid and only 30% remained as glutamic acid. TABLE MOLECULAR

WEIGHTY

Method SDSlpolyacrylamide Phosphate buffer 5% 11%

15% Tris buffer 6.5% Sephadex G50 Amino acid analysis

II

OF INITIATION

FACTORP

IF1

IF2a

IF3

gels -

117,000

8,900

-

8,200

-

22,200 22,800

9,300 8,700

118,000 -

-

a The values from the SDWpolyacrylamide gel method are derived from the data reported in Fig. 6. Other values are cited in the text.

PROTEIN

SYNTHESIS TABLE

AMINO

ACID

Tryptophan Total number dues

of resi-

5.1 2.1 6.4 7.3 7.0 3.2 7.0 2.6 5.2 2.4 7.5 2.7 5.0 6.4 2.0 2.0 0.36 0.0” 0.1 74.9

IFl,

IF2,

AND

IF3

FROM

weights and both stimulate plex formation.

III

IF1 Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Cysteine

FACTORS

COMPOSITIONS”

Number

Residue

INITIATION

of residues IF2a 83 19 83 95 47 54 152 30 79 130 102 23 54 66 14 17 6b 2 1056

IF3 23.1 1.2 16.4 12.7 6.2 8.9 36.5 8.5 15.3 11.3 15.9 5.2 12.6 16.7 3.4 5.5 1.7b 1.3’ 0.1 201.2

(1 Details of the procedures and calculations are given in the text. At least two different preparations of each of the purified initiation factors were analyzed. The values for each residue are the average of six determinations except for valine, isoleucine, serine, threonine, and tryptophan, where only two determinations were made. b, c Cysteine was determined either as cysteic acid (denoted in the Table with the superscript b) or with Ellman’s reagent (13) in the absence of urea (denoted with the superscript c).

Therefore the analyses of these amino acids reported here were carried out with samples free of glycerol. The analyses show that all three factors contain high amounts of charged amino acid residues and lower amounts of hydrophobic residues. The low levels of tyrosine and tryptophan are consistent with the small extinction coefficients determined at 280 nm for these proteins (see below). The compositions are different from those reported for ribosomal proteins (39). The composition of IF1 from E. coli strain Q13 was determined previously by Lee-Huang et al. (21) and is strikingly different from the one reported here. We conclude that the two IF1 proteins analyzed are different although they have similar molecular

E. coli

initiation

635

com-

Determination of protein concentrations. The protein concentration of puri-

fied factors was determined by three independent methods: (i) calorimetrically by the procedure of Lowry et al. (20); (ii) spectrophotometrically by measuring absorbance at 280 and 260 nm (40); and (iii) from total recovery of amino acid residues. The method of Lowry et al. (20) gave results in close agreement with the amino acid analysis data. Absorbance values at 280 nm for the three initiation factors were lower than expected for a typical protein. Values of 0.30 + 0.05 were routinely observed for solutions of each of the factors at 1 mg/ml, measured according to the Lowry method. Based on the amino acid compositions shown in Table III and on molar extinction coefficients at 280 nm of 14,700 and 5,600 cm-’ for tyrosine and tryptophan, respectively, theoretical absorbance values for 0.1% solutions were calculated: for IFl, 0.34; for IF2, 0.26; and for IF3, 0.20. The theoretical values are close to those actually observed. Because of the presence of 2-mercaptoethanol in the buffers routinely used, absorbance values for the factors were not used for quantitation. The method of Lowry was used routinely for accurate measurements. Biological

Properties

The highly purified initiation factors were assayed for their ability to stimulate protein synthesis or various partial reactions of the initiation process. Protein synthesis was measured in a system which utilized 70 S ribosomes washed in high salt, an SlOO supernatant fraction, and phage R17 RNA as the messenger. The partial reactions measured formylmethionyl-tRNA binding to 30 S ribosomal subunits in the presence of A-U-G, or to washed 70 S ribosomes with either A-U-G or phage R17 RNA. Formylmethionylpuromycin synthesis was also studied by using phage R17 RNA as messenger. Saturating amounts of the three initiation factors were added in various combinations to the assay systems and the stimulations obtained are reported in Table IV.

636

ET AL.

HERSHEY TABLE FACTOR Additions

REQUIREMENTS Formylmethionyl-tRNA

30 S A-U-G None IF1 IF2 IF3 IF1 IF1 IF2 IF1

+ + + +

IF2 IF3 IF3 IF2

+ IF3

71 69 1747 71 3257 59 1117 1178

IV

IN ASSAYS FOR INITIATION”

70 S A-U-G 544 467 1239 486 1898 382 1457 2311

binding 70 S R17 RNA 94 106 226 94 353 128 591 837

Formylmethionylpuromycin synthesis R17 RNA 62 48 95 78 288 99 1084 1308

Pr&A;i;ynR17 RNA 259 403 359 1438 389 1505 1520 1951

(1 The amounts of factors added are: IFl, 0.20 pg; IF2a, 2.4 pg; IF3, 0.45 pg. The formylmethionyl-tRNA binding assays were performed as described under Experimental Procedures except for those reported in the first column, where 30 S subunits (0.2Azw units), prepared as described by Fakunding and Hershey (91, were used in place of 70 S ribosomes. The assay for formylmethionylpuromycin synthesis was carried out with the same ingredients used for formylmethionyl-tRNA binding reported in the third column, except that 0.1 mM puromycin was included in the reaction mixture and the product was analyzed by the procedure of Leder and Bursztyn (42). Protein synthesis was assayed in 50-~1 reaction mixtures containing: 30 mM Tris-HCl, pH 7.5, 5 mM Mg-acetate, 70 mM NH&l, 1 mM dithioerythritol, 0.02 mM each of 19 nonradioactive amino acids plus [3H]leucine (specific activity 30 Ci/mol), 1 mM ATP, 0.2 mM GTP, 0.6 mM folinic acid, 12 mM phosphocreatine, 2 units of creatine phosphokinase, 40 pg of unfractionated tRNA, 15 pg of R17 phage RNA, 0.5 A,,, units of high salt-washed 70 S ribosomes, 12 ~1 of dialyzed SlOO supernatant fraction, and factors as indicated. The mixtures were incubated 30 min at 3O”C, and [3H]leucine incorporation was determined by hot trichloroacetic acid precipitation and filtration. The results of all assays are reported as counts per minute following subtraction of a background of 50 cpm.

In the simplest reaction, the binding of formylmethionyl-tRNA to 30 S ribosomal subunits, IF2 is clearly the most important factor. When each factor was tested separately, only IF2 caused significant stimulation of binding. IF1 stimulated IF2 activity nearly twofold, but IF3 caused inhibition of formylmethionyl-tRNA binding, in agreement with the results of other workers (41). A somewhat different pattern of stimulation was seen when 70 S ribosomes were used with A-U-G. IF2 was again critical, but the extent of IF1 stimulation was reduced while IF3 stimulated to the same extent as IFl. When lesser amounts of IF2 are used in the assay system, for example as reported in Fig. 2 for IF1 activity, formylmethionyl-tRNA binding becomes more dependent on either IF1 or IF3, and up to lo-fold stimulation by either factor has been observed. When phage R17 RNA was used as the messenger in the formylmethionyl-tRNA binding assay system, a more pronounced dependence on all three factors was seen. IF2 was again essential, either IF1 or IF3 stimulated IF2 activity,

and all three factors gave maximal stimulation. By using phage R17 RNA as messenger, it was possible to measure protein synthesis in a system dependent on initiation factors. However, IF3 alone was strongly stimulator-y and rather little dependence on IF1 or IF2 was obtained. It is likely that IF1 and IF2 are contaminants in the SlOO supernatant fraction. A satisfactory protein synthesis assay system requires that the elongation and termination factors and aminoacyl-tRNA synthetases be prepared free of initiation factors. In order to circumvent the requirement for the SlOO supernatant fractions, formylmethionylpuromytin synthesis was studied with phage R17 RNA. This reaction is analogous to the formation of the first peptide bond and is therefore useful in determining whether bound formylmethionyl-tRNA is in a reactive state. Strong dependence on both IF2 and IF3 was seen, while IF1 stimulated the system only slightly. The various assays help establish that IF2 and IF3 are required for initiation of protein synthesis.

PROTEIN

SYNTHESIS

INITIATION

FAC TORS IFI, IF2, AND IF3 FROM E. coli

The role of IF1 is less clear, however. It is not yet established that IFl, IF2, and IF3 are the only initiation factors and that their presence is sufficient to obtain maximal rates of initiation of protein synthesis. ACKNOWLEDGMENTS We thank E. Beckman for performing the acid analyses, W. F. Benisek, S. Langberg, Benne for critically reading the manuscript, Bittick and P. Giese for expert typing of the script. This work was supported by Grant from the American Cancer Society.

amino and R. and C. manuNP-70

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