[6] Analysis of initiation factor function in highly fractionated and unfractionated reticulocyte lysate systems

[6]

ASSEMBLY

OF THE

EUKARYOT1C

INITIATION

COMPLEX

61

this complex is bound to 40 S or 80 S ribosomes and thus facilitates the recycling of EIF-I • Co-EIF-I during peptide chain initiation."' In analogy with the known protein synthesis initiation mechanism in Escherichia coil, we propose that the release of the initiation factors and GTP hydrolysis occur during the joining of the ribosomal subunits at the last stage of the 80 S initiation complex formation. t0 A. Majumdar, R. Roy, A. Das, A. Dasgupta, and N. K. Gupta, Biochem. Biophys. Res. Commun. 77, 161 (1977).

[6] A n a l y s i s o f I n i t i a t i o n F a c t o r F u n c t i o n in Highly Fractionated and Unfractionated Reticulocyte Lysate Systems

By

BRIAN SAFER, ROSEMARY JAGUS,

and WAYNE M. KEMPER

As early as 1972, the basic sequence of events occurring during assembly of the 80 S initiation complex had been formulated (Fig. I). ~-3 Nine polypeptide initiation factors required for this process have since been purified to homogeneity? -~ A summary of recently proposed initiation factor functions is presented in Table I. Although details of individual factor function have been studied extensively in highly fractionated systems, 1°-]" confirmation of individual initiation factor function in complex systems is incomplete. This corroboration is necessary, however, because of uncertainties resulting from the low activities of all reconstituted systems (less than 5% of unfractionated systems), the inability of most purified translational components to actively recycle in these sysM. H. Schreier and T. Staehelin, Nature (London), New Biol. 242, 35 (1973). C. H. Darnbrough, S. Legon, T. Hunt, and R. J. Jackson, J. Mol. Biol. 76, 379 (1973). :~D. H. Levin, D. Kyner, and G. Acs, J. Biol. Chem. 248, 6416 (1973). 4 B. Safer, W. F. Anderson, and W. C. Merrick, J. Biol. Chem. 250, 9067 (1975). M. H. Schreier, B. Erni, and T. Staehelin, J. Mot. Biol. 116, 727 (1977). ~ W. C. Merrick, W. M. Kemper, and W. F. Anderson, J. Biol. Chem. 250, 556 (1975). 7 W. M. Kemper, K. W. Berry, and W. C. Merrick, J. Biol. Chem. 251, 5551 (1976). W. C. Merrick and W. F. Anderson, J. Biol. Chem. 250, 1107 (1975). 9 R. Benne and J. W. B. Hershey, Proc. Natl. Acad. Sci. U.S.A. 73, 3005 (1976). ~o B. Safer, D. Peterson, and W. C. Merrick, in '.'Translation of Synthetic and Natural Polynucleotides" (A. B. Legocki, ed.), p. 24. Publ. House Poznan Agricultural University, Poznan, Poland, 1977. H H. Trachsel, B. Erni, M. H. Schreier, and T. Staehelin, J. Mol. Biol. 116, 755 (1977). ~ R. Benne, M. Luedi, and J. W. B. Hershey, J. Biol. Chem., in press (1978).

METHODS IN ENZYMOLOGY, VOL. LX

Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. 1SBN 0-12-181960-4

62

INITIATION OF PROTEIN SYNTHESIS

"~- ~

~ J/"

( ~ ~ UAC 8

UAC

2.BINDINGOF INITIATOR Met-tRNAfJ

60S 1.FORMATIONOF NATIVE40S RIBOSOMAL BUBUNITS

mRNA AUG

, POLY(A) 3.BINDINGOF mRNA

Met

Met

80B

[6]

' POLY(A) U]C !

~

S

~l,

3

/

4.60SRIBOSOMAL SUBUNITJOINING

80 S INITIATION COMPLEX

FIG. l. Schematic representation of the four basic steps of eukaryotic protein synthesis initiation. The specific roles that initiation factors play in this assembly process are summarized in Table I.

tems, and the loss of physiological control mechanisms present in unfractionated lysate and the cell. Determination of the initiation factor, tRNA, mRNA, and nucleotide composition of preinitiation complexes and the distribution of translational components in both unfractionated and reconstituted systems has been complicated by the instability of these complexes. This has resulted in an apparent inefficient utilization of various translational components, as well as uncertainties in the requirements for formation and composition of intermediates formed during assembly of the 80 S initiation complex. Additional problems are introduced, however, by the use of cross-linking ~:eagents to stabilize these complexes, since artificial associations of translational components can easily occur. We have found that exchange of the nonhydrolyzable GTP analog, GDPNP, for ribosomal bound GTP, or the presence of GTP-regenerating systems, permits highly efficient and stable binding of RNA and initiation factors during sucrose density gradient analysis without the use of chemical fixation. 1oRecent modifications in standard sucrose density gradient techniques, polyacrylamide gel electrophoretic analysis and sample preparation, aminoacyl-tRNA and mRNA analysis, rapid procedures for eukaryotic initiation factor preparation, and radiolabeling techniques which do not alter the functional activities of most initiation factors, now permit exam-

[6]

ASSEMBLYOF THE EUKARYOTIC INITIATION COMPLEX

63

TABLE I MOLECULAR WEIGHTS AND PROPOSED FUNCTIONS OF EUKARYOTIC INITIATION FACTORS

Molecular weight

Nomenclature New

Old

SDS G e l s

Native

Primary function

15,000

15,000

Preinitiation complex stabilization; 80 S Ribosomal Dissociation

125,000

Met-tRNAfbinding to native 40 S [Regulationof Met-tRNAy binding]<`

elF-I

elF-2

IF-MP

elF-2A

IF-MI

55,000 52,000 35,000 65,000

65,000

f 130,000

[ 110,000

eIF-4A IF-M4 elF-4B IF-M3 elF-4C IF-M2B/3

69,000 49,000 43,000 39,000 35,000 50,000 80,000 19,000

elF-4D

IF-M2Ba

17,000

15,000

elF-5

IF-M2A

150,000

125,000

elF-3

IF-M5

| ~ | |

700,000

50,000 80,000 17,000

Formation of native 40 S; mRNA binding mRNA binding mRNA binding Preinitiation complex stabilization; subunit joining [Preinitiation complex stabilization|" Initiation factor release; subunit joining

Exact function uncertain.

ination of the specific functions of initiation factors in the unfractionated reticulocyte lysate. Materials

General L-[14C]Valine (260 mCi/mmol), [14C]formaldehyde (40 mCi/mmol) from New England Nuclear; L-[35S]methionine (800-1200 Ci/mmol) and L-[3H] methionine (0.5-2 C i / m m o l ) f r o m Amersham Corp.; [5'-:~H]polyuridylic acid (500/~Ci/p~mol P) from Miles Research Products; phenylhydrazine, heroin, tris(hydroxymethyl)aminomethane (Tris), pancreatic ribonuclease

64

INITIATION OF PROTEIN SYNTHESIS

[6]

A from Sigma; sucrose (ultrapure, ribonuclease free) from Schwarz Bioresearch, Inc.; ATP, GTP, phosphoenol pyruvate, creatine phosphate (dipotassium salt), sodium heparin, free amino acids, creatine phosphokinase (200 IU/mg), pyruvate kinase (50 IU/mg) from Calbiochem; guanylyl imidodiphosphate (GDPNP) and crude yeast ribonucleic acid from B0ehringer Mannheim Biochemicals; NCS tissue solubilizer from Amersham Corp.; ethylene glycol, trichloroacetic acid, cetyltrimethylammonium bromide, type GF/C Whatman glass fiber filters, sodium borohydride from the A. H. Thomas Company; nitrocellulose filters (type HA, 0.45/~m pore size, 25 mm diameter) from the Millipore Corporation; Sephadex G-25 and Sepharose CL-4B from Pharmacia Fine Chemical~, Inc.; Hydromix and LSC scintillation cocktail solutions from Yorktown Research; oligo(dT)-cellulose from Collaborative Research, Inc.; acrylamide, bisacrylamide, TEMED, and sodium dodecyl sulfate from BioRad Laboratories; and X-Omat XR-2 film from Kodak. B. S o l u t i o n s a n d R e a g e n t s

Solution A: 140 mM NaCI, 5 mM KCI, 7.5 mM Mg acetate, autoclaved Solution B: 200 mM Tris. HCI, pH 7.5, 1.7 M KC1, 50 mM Mg acetate stored in small aliquots at --20 °, used once, and discarded Solution C: 20 mM HEPES.HCi, pH 7.5, 100 mM KCI, 1 mM dithiothreitol, 0.1 mM EDTA, 10% glycerol Solution D: same as solution C, except that Tris replaces HEPES Phenylhydrazine hydrochloride 2.5%: 25 g are dissolved in 1 liter of water (deoxygenated by bubbling No) and neutralized to pH 7 with 1 N NaOH. Aliquots are stored protected against light in airtight containers at --20% used once, and discarded 19 Minus radiolabeled amino acid mixture prepared as described by Darnbrough et al. z Creatine phosphokinase: dissolve lyophilized enzyme in 50% glycerol (v/v) to 10 mg/ml and store at - 2 0 ° Heroin: a 1 mM solution in ethylene glycol prepared by adding 65 mg to 2 ml of 100 mM KOH. Sufficient 100 mM Tris .HCI is added to neutralize to pH 7.8-8.2, and the volume is brought up to 20 ml; 80 ml of ethylene glycol are then added, the solution is clarified by centrifugation, and stored at - 2 0 ° 20 X SSC: 6 M NaC1, 0.6 M sodium citrate, 0.2 M Tris pH 7.2 autoclaved SDS: 4% sodium dodecyl sulfate RNase: 20/zg of ribonuclease A per milliliter of 0.5 M NaC1, I0 mM magnesium acetate, 10 mM Tris-HC1, pH 7.2, freshly prepared

[6]

A S S E M B L Y OF T H E E U K A R Y O T I C I N I T I A T I O N C O M P L E X

65

3% CTAB: 3 g of cetyltrimethylammonium bromide per 100 ml of 3 mM sodium acetate, pH 5.2 0.1% RNA: 100 mg of unfractionated yeast RNA per 100 ml of 0.5 M sodium acetate, pH 5.2 [5'-3H]Polyuridylate: 0.625/~g/ml in sterilized water. Use 50/zl ~ 4 × 104 cpm per assay Procedures

Preparation of Lysate New Zealand White female rabbits (2-3 kg) are injected subcutaneously (interscapular region) with 0.25 ml of phenylhydrazine per kilogram (2.5% w/v) for 5 days. A reticulocyte count of 70-80% and hematocrit of 20% should be achieved by day 5. Vitamin B12 and folic acid supplementation are not required. For protein synthetic activity, the rabbits are bled by cardiac puncture on day 8 with a 60-ml disposable syringe containing 0.1 ml of heparin (500 U/ml) and a No. 14 Huberpoint needle. A higher yield of initiation factors is obtained by bleeding on day 7, but translational activity (normalized to ribosome content) is approximately 30% lower. Immediately preceding sacrifice, rabbits are anesthetized individually by intraperiton~al injection of 30 mg pentobarbital. For optimal translational activity, hypoxic blood should not be utilized. The reticulocytes are collected and washed 3 times with 4 volumes of buffer A to remove serum proteins and heparin. The cells are pooled after the first wash, and the buffy coat is carefully removed by aspiration after the second wash to minimize RNase contamination. It is also important to carefully remove as much buffer A as possible after the third wash to reduce carryover of salt. During the washing procedure, cells are collected by centrifugation for 10 rain at 4000g, all procedures are conducted at 2°. Cells are lysed in 1.5 volumes of water per volume of packed cells, and yields are maximized by stirring for 5 min. Cell membranes and mitochondria are removed by centrifugation for 20 min at 16,000 g at 2°. The supernatant is carefully decanted into a separate container to ensure homogeneity, immediately frozen in 1-ml aliquots at --80 °, and stored at the vapor temperature of liquid nitrogen. All solutions and equipment are sterilized, and standard precautions are taken to eliminate RNase contamination. Approximately 200 ml of lysate are obtained fi-om 10 rabbits.

Characterization of the Lysate Preparations Before utilization, reticulocyte lysate preparations are evaluated for translational activity, ribosome content, and translational regulation by heroin.

66

I N I T I A T I O N OF P R O T E I N S Y N T H E S I S

[6]

Assay Conditions for Translation. To partially thawed 1-ml aliquots of reticulocyte lysate, 10/~1 of creatine phosphokinase and 25/xl of hemin are added and carefully mixed; 5/zl each of 2 M KC1 + 10 mM Mg(OAc)2,200 mM creatine phosphate, 19 minus radiolabeled amino acid solution, and radiolabeled amino acid are then added per 80/zl of reticulocyte lysate. It is not generally required that K ÷ and Mg ~'+ concentrations be adjusted more finely. The final volume of 100/21 may be increased up to 110/xl by addition of other solutions without greatly affecting translational activity. To measure the rate of amino acid incorporation, 5-~i aliquots of the incubation mixture are added to approximately 1 ml of water. Pipetting systems with disposable plastic tips are most convenient, and it is important to rinse these 1 or 2 times for quantitative delivery of samples. An equal volume of 10% trichloroacetic acid (w/v) is then added. When short incubation times are studied, it is important to deacylate the aminoacyi-tRNA pool. This is done by incubating for 20 min at 90° and cooling on ice, before plating on either nitrocellulose or glass fiber filters with 5% TCA. Filters are dried and counted by standard liquid scintillation techniques. Quench correction with lac- or '~S-labeled radionuclides is generally not required. As a general precaution, unused remaining aliquots of lysate and creatine phosphate are discarded. Radionuclides are stored at - 140°, and all other components are stored at --20 °. When incubated at 30° in the presence of hemin, synthesis of hemoglobin is linear for more than 40 min. In the absence of heroin, normal rates of protein synthesis are maintained for 4-6 min, but then translational activity abruptly becomes inhibited by greater than 90%. Failure to maintain linear rates of amino acid incorporation for 40 min or failure of translation to become inhibited in the absence of hemin generally indicates a low absolute activity, and such lysates are not used for functional studies. Initiation factors prepared from these, however, appear to be normal. Determination of the Rate of GIobin Synthesis. To determine the rate of protein synthesis, or the pool sizes of some translational components accurately, it is important to know the exact specific activity of the radionuclide pool. In the case of amino acids, this may be done most simply and quickly by measuring the amount of radioactivity incorporated in hot TCA-precipitable translational products. Two or three levels of radiolabeled amino acid, sufficient cold amino acid, and short incubation times (10-20 rain) are used to ensure that synthesis does not become limited. The specific activity and size of the amino acid pool may be obtained using the following relationship: [a/(a +X)] • b = Y where a = pmol radioactive amino acid; b = initial specific activity of the

[6]

ASSEMBLY OF THE EUKARYOTIC INITIATION COMPLEX

67

radiolabeled amino acid; X = pmol of endogenous amino acid; Y = disintegrations per minute per picomole of product. By using different input levels of the radioactive amino acid, a series of simultaneous equations may be generated and solved. Based on the amino acid composition of the translated product, the absolute rate of translation can be calculated. In the case of endogenous mRNA, the use of [aaS]methionine or [14C]lysine is advantageous. Specific activities in excess of 1000 Ci/mmol may be obtained for [aaS]methionine (Amersham/Searle), and both methionine and lysine have equal numbers of amino acid residues per completed a- or /3-globin chain (1 and 12, respectively). Estimation of Ribosomal Content. To compare translational activities of different lysate preparations, it is necessary to express the rate ofglobin synthesis per unit ribosomal content. The easiest method we have found, which avoids possible errors obtained by gel filtration or pelleting of ribosomes, is to measure the total ribosomal A~60 following sucrose density gradient analysis. This requires that ribosomal populations be sufficiently resolved from hemoglobin and other low-molecular-weight components left on the top of the gradients, but that large polysomes not be pelleted. Centrifugation of the reticulocyte lysate following hemin deficiency translational inhibition is convenient since all ribosomal particles are present only as 40 S, 60 S, 80 S, or 100 S species, free of mRNA or tRNA. Analysis of the total ribosomal population from the AzG0 profile using standard weighing procedures is sufficient for most pu.rposes; 160-240 pmol of 80 S equivalents per milliliter of lysate are generally obtained.

Initiation Factor Purification A rapid procedure for the purification of initiation factors essential for assembly of the 80 S initiation complex is presented by Goldstein and Safer in this volume [12]. For the purpose of initiation factor identification in unfractionated systems, it is not necessary to have the factors as purified as when quantitative information is required. For example, with partially purified preparations of eIF-3 and eIF-2, only true eIF-3 or -2 subunits (determined by analysis of homogeneous preparations) are bound to 40 S ribosomal subunits, while contaminating subunits are separated during centrifugation. Methods for the purification of the major initiation factors to near or apparent homogeneity are: eIF-2, 4 eIF-3, 9 eIF-4A, la eIF-4B? eIF-4C, 7 and eIF-56 (also see Merrick, this volume [8]). 1:~B. Safer, S. L. Adams, W. M. Kemper, K. W. Berry, M. Lloyd, and W. C. Merrick, Proc. Natl. Acad. Sci. U.S.A. 73, 2584 (1976).

68

I N I T I A T I O N OF P R O T E I N SYNTHESIS

[6]

Preparation of Radiolabeled Initiation Factors Reductive Methylation of Initiation Factors. Initiation factor labeling by reductive methylation is modified from the procedure of Ottesen and Svenson. 14 Initiation factors at a protein concentration of 1-5 mg/ml are dialyzed against solution C to remove Tris buffer utilized during purification procedures; 1 M potassium borate buffer, pH 9.5, is then added to a final concentration of 100 mM. [~ac]- or ['~H]formaldehyde, available as 1% aqueous solutions, is added to a final concentration of 1-5 raM. The optimal concentration of formaldehyde to use varies with the different reactivities of initiation factors, and is determined by functional analysis of the radiolabeled factor. Freshly prepared 25 mM sodium borohydride is then added in l0 equal increments at 3-min intervals. The labeled factors are then rapidly separated from free formaldehyde by Sephadex G-25 chromatography. The appearance of the radiolabeled protein is monitored by liquid scintillation counting using Hydromix. To obtain good yields of labeled factors at high protein concentrations, the pooled column fractions are dialyzed against saturated (NH4)~SOa, collected by centrifugation, dissolved in and extensively dialyzed against solution D. All operations are conducted at 2-4 °. All factors are tested for functional activity before and after reductive methylation. Although the specific activity of initiation factors labeled by reductive methylation is lower than that attainable by iodination or phosphorylation, there are two important advantages: (i) better physical and biological stability of the radionuclide and labeled factor, and (2) generation of relatively large amounts of labeled factors. These conditions minimize the selective utilization of nonrepresentative factor populations. Testing of Radiolabeled Initiation Factors. Since translation of natural mRNA in the fractionated reticulocyte system is absolutely dependent on the six factors referenced above in the section Initiation Factor Purification, their activities can be tested by titration of the single missing component. Since it is difficult to prepare and test all components required, alternative, less complex methods of testing for functional activity are desirable. The AUG-dependent methionylpuromycin model assay system offers a way to test for functional activity ofeIF-2, elF-3, eIF-4C, and eIF-5 using readily obtainable, commercially available reagents; alternatively, eIF-2 activity may be assessed directly by nitrocellulose filter retention of the [eIF-2.Met-tRNAf.GTP] ternary complex (see Goldstein and Safer, this volume [12]). The accumulation of approximately equivalent amounts of these labeled initiation factors with equimolar levels of Met-tRNAr and ~4 M. Ottesen and B. Svenson, C. R. Trav. Lab. Carlsberg 38, 445 (1971).

[6]

ASSEMBLYOF THE EUKARYOTIC INITIATION COMPLEX

69

mRNA in a 48 S preinitiation complex formed during edeine inhibition may also be a test of functional equality with the endogenous initiation factor pool (see below, General Comments, entry 6). Globin m R N A Purification

Salt-washed reticulocyte polysomes are dissolved in 500 m M N a C l 0.5% S D S - 2 0 m M T r i s . H C l , pH 7.5, buffer to a final A2~,~ of 20 per milliliter. The temperature is raised to 60% and the solution is passed through an oligo(dT)-cellulose column at a flow rate of 2-3 ml/min. Approximately 1 g of oligo(dT)-cellulose is required per 10'~ A.,~0 of ribosomes. Poly(A)-bound material is washed with 10-20 column volumes of 600 m M NaC1-100 m M Tris. HCI, pH 7.5, until all traces of SDS are removed. RNA is eluted with HzO, concentrated by precipitation with 2 volumes of ethanol at - 2 0 ° overnight, and collected by centifugation at - 2 0 ° at 10,000 g for 20 rain. At this stage of purification, globin mRNA sedimenting at 10 S is approximately 40% pure, the remaining RNA consisting of 17-18 S and 28 S species, some of which appear to contain aggregated mRNA. Additional purification can be obtained by molecular-sieve chromatography. ~:' Approximately 500 A 26oof oligo(dT)-cellulose-prepared mRN A is applied to a 5 × 100 cm Sepharose CL-4B column equilibrated with 500 m M K C I - I m M DTT and 100 m M Tris.HCI, pH 7.5. Highly purified 10 S globin mRNA free of ribosomal RNA contamination is obtained at a Ve/Vt of 0.5. The RNA of this peak is pooled and concentrated as described above and is dissolved in water to a final concentration of20A 2~.)per milliliter. The yield of 10 S globin mRNA purified in this manner is approximately 0.6% of the initial polysomal A,,60. R i b o s o m a l Subunit Preparation

40 S and 60 S ribosomal subunits were prepared by puromycin dissociation according to established procedures. '~ Aminoacylation o f t R N A f Met

Met-tRNAf was prepared by aminoacylation of unfractionated rabbit liver tRNA using E. coli synthetase as previously described. '7 ,5 M. Zeichner and R. Stern, Biochemistry 16, 1378 (1977). 'n G. BIobel and D. Sabatini, Proc. Natl. Acad. Sci. U.S.A. 68, 390 (1971). ,r W. M. Stanley, Jr., Anal. Biochem. 48, 202 (1972),

70

INITIATION OF PROTEIN SYNTHESIS

[6]

Sucrose Density Gradient Analysis of Initiation Factor, mRNA and Aminoacyl-tRNA Distribution in Fractionated and Unfractionated Reticulocyte Lysate

General Comments

The major advantages and disadvantages of studying initiation factor function in highly fractionated systems and the unfractionated reticulocyte lysate are compared: Fractionated systems

Lysate

1. The exact amounts and types of translational components are determined by experimental design and availability. 2. Various possible intermediary complexes formed during initiation complex assembly can be readily obtained by omission of specific components.

1. The composition of the lysate is fixed. Additional components can be easily added, however. 2. The type, distribution, and composition of various preinitiation complexes are determined by the physiological state of the translational apparatus. Physical removal of specific lysate components is generally not possible. Functional removal of specific substances through the use of various inhibitors, antibodies, etc., however, can be used to great advantage. 3. Pool sizes of endogenous lysate components must be determined for quantitative analysis using radiolabeled components. 4. Zone sedimentation of lysate on sucrose gradients must be used to resolve discrete ribosomal populations. Nitrocellulose filter binding cannot be used since all ribosomal species, as well as both free and bound initiation factors, are retained. While gel filtration is capable of rapidly separating free and bound initiation factors, resolution of ribosomal subunits and monomers is inadequate for most purposes. Direct analysis of the protein and RNA composition of sucrose density gradient fractions is complicated by the relatively low concentrations of translational components in reticulocyte lysate. Direct analysis, as well as use of specific radiolabeled factors, are possible, however.

3. The specific activities of radiolabeled components are known.

4. Depending on experimental design, free translational components may be separated from those bound to intermediary preinitiation complexes by rapid simple procedures, such as molecular sieving or binding to nitrocellulose filters.

5. Direct polyacrylamide gel electrophoretic analysis of protein and mRNA distribution is possible, since the pool sizes can be experimentally determined.

[6]

ASSEMBLY OF THE EUKARYOTIC INITIATION COMPLEX

6. The activity of most fractionated translation systems is less than 5% of in vivo rate of protein synthesis. 7. Catalytic recycling of most initiation factors cannot be demonstrated. 8. Physiologic regulatory mechanisms, such as that described for the regulation of globin synthesis by heroin, are largely, if not totally, lost.

71

6. The activity of the unfractionated reticulocyte lysate is usually 70-100% of the rate in intact cells. 7. Pool sizes of most translational components are small relative to their turnover numbers. 8. Translational control mechanisms generally appear intact.

In general, sucrose density gradient analysis of fractionated translational components does not require greater resolution than that obtained using a SW 56 rotor. Polysome formation does not usually occur, while high binding efficiencies of assay components allow adequate resolution of free and bound components. In the unfractionated reticulocyte lysate, the sedimentation value of ribosomal components ranges from 43 S to approximately 200 S; in addition, large pools of unbound components and released translation products make resolution of components bound to the small ribosomal subunit more difficult. For these reasons, longer sucrose gradients, such as those obtainable using Beckman SW 41 or SW 27 rotors (when larger volumes must be analyzed), are preferable.

Conditions for Sucrose Density Gradient Analysis Linear 10 to 35% sucrose gradients of the following ionic composition are satisfactory for use with both fractionated and unfractionated systems: 10 mM Tris. HC1, pH 7.5; 85 mM KCI; 2.5 mM Mg(OAc)~; increased complex stability in fractionated systems may be obtained by utilization of 5 mM MgC12. Sucrose solutions are freshly prepared from frozen aliquots of 20 X buffer stocks (solution B), sucrose and autoclaved glass-distilled water. Gradients are formed several hours before use and left to stabilize at 4 °"

Initiation complex formation using highly purified components is conveniently performed in 100-/xl reaction cocktails containing 10-40 pmol each of 40 S and/or 60 S ribosomal subunits, [3H]Met-tRNAf (purified or unfractionated), AUG (0.3 A.,~0 unit) or globin mRNA, initiation factors (see Table I for functional molecular weights) and the following reagents: 10 mM Tris • HCI, pH 7.5; 85 mM KCI; 2.5 mM Mg(OAc)2, 0.08 mM GTP-4 mM PEP-5 U pyruvate kinase or 0.8 mM GDPNP; 0.5 mM ATP. Met-

72

INITIATION OF PROTEIN SYNTHESIS

[6]

tRNAf binding to 40 S ribosomal subunits for subsequent 80 S initiation complex formation with natural mRNA is absolutely dependent on eIF-2, and appears to be stabilized by eIF-3. Binding of mRNA to the [40 SMet-tRNAf.eIF-2.eIF-3] preinitiation complex is promoted by elF-4A, eIF-4B, eIF-4C, and ATP. Initiation factor release and 60 S ribosomal subunit joining require eIF-5-mediated GTP hydrolysis. With highly purified components, the sequence of component addition to the incubation cocktail is not important (with one exception, see p. 74). Samples are incubated at 30° for l0 rain, chilled and layered on sucrose gradients without dilution, and centrifuged at 4 ° in a Beckman SW 56 rotor at 55,000 rpm for 105 rain (80 S binding) or 150 rain (40 S binding). The 3.8-ml gradients are fractionated using an ISCO Universal flow cell, Model UA-5 absorbance monitor with a type 6 optical unit and a Bio-Rad fraction collector. Gradients are displaced upward with 60% sucrose, and absorbance is monitored at 254 nm. Superior resolution is obtained using this bulk flow analysis system compared to other laminar flow systems we have used. The amount of unfractionated reticulocyte lysate used for sucrose density gradient analysis will depend largely on the component being analyzed and the experimental objectives. With currently available ['%S]methionine preparations, final specific activities in excess of l0 s dpm per picomole of methionine can be obtained, and 50-200 /A of lysate are sufficient for most purposes. Approximately 100 p.I of lysate are required for hybridization assays of [5'-:~H]polyuridylic acid (specific activities of -106 dpm per picomole of nucleoside residue) to the 3'-polyadenylate tracts of globin mRNA. The direct identification of preinitiation complex components, such as initiation factors, by polyacrylamide gel electrophoresis (PAGE) techniques generally requires 1-4 ml oflysate; the use of radiolabeled initiation factors reduces this by one order of magnitude, but introduces uncertainties with respect to functional identity. In general, we have found that superior resolution of preinitiation complexes is obtained by minimizing the number of individual sucrose gradients pooled to obtain sufficient material for analysis, by using larger sucrose gradients. For purposes of roughly estimating the amount of lysate required for analysis of ribosomal bound components, the average reticulocyte lysate preparation will contain approximately 200 pmol of equivalent 80 S ribosomal couples per milliliter. The free small and large ribosomal subunit pools will range from 0.5 to 2 pmol/ml. The use of specific inhibitors of protein synthesis can increase or decrease the size of the ribosomal subunit pools by 5- to 10-fold. While various inhibitors of protein initiation may result in the total elimination ofpolysomes, most of the ribosomal subunits released reassociate under physiological conditions to form nonfunctional

[6]

73

ASSEMBLY OF THE EUKARYOTIC INITIATION COMPLEX

80 S ribosomal couples. Details of gradient conditions will be presented in the figure legends used to illustrate the study of Met-tRNAf, globin mRN A, and initiation factor distribution in the unfractionated reticulocyte lysate. For optimal resolution of ribosomal populations from lysate, a minimal 1:1 dilution is required. Generally, reaction cocktails are diluted into 2 volumes of ice cold 10/xM GDPNP, which effectively stops further activity of the lysate by rapidly lowering the temperature, inhibiting GTPdependent processes, such as elongation and factor release, and establishing unfavorable ionic conditions. The sedimentation coefficients of ribosomal subunits are always higher in lysate than in fractionated systems because of the association of nonribosomal proteins. The resolution obtained in these various systems and the approximate sedimentation coefficients of ribosomal particles in normal lysate are presented in Fig. 2A and C. Note that "halfmers" consisting of small ribosomal subunits as-

A

0.8 I ]

T

0.6

ww _.1 _.1 cl~ llo <<

~_ ~ ~u LUUJ ao<

II

CONTROL

6OS

B

0.4E

0.4

0.2

6z ~

8 C

~_

÷ EDEINE

16

D

CONTROL

24

30

+ EDEINE

0.6

I

8OS

m

~,

0.4

5

0.2

60S

6

12

/

,3s

!

18

24

l 75SA

l//

30

[~

/ \

36 6 FRACTION NUMBER

12

18

24

30

36

FIG. 2. Resolution of ribosomal populations by sucrose density-gradient centrifugation. Standard 100-/zl rabbit reticulocyte lysate reaction mixtures were incubated at 30° for 8 rain. Edeine was added to (B) and (D) at 4 min. Samples were analyzed by sucrose density-gradient centrifugation ( 15 to 35% linear gradients) in Beckman SW 41 rotors for 75 rain (A and B) or 4.5 hr (C and D). In (A) and (B), both cetyltrimethylammonium bromide (CTAB) and hot trichloroacetic acid (TCA)-precipitable [34S]methionine distribution were determined in duplicate gradients. The specific activity of the [:~:'S]methionine pool was 120,000 dpm/pmol. In the presence ofedeine, Met-tRNAr binding to the small ribosomal subunit is increased 6-fold and the sedimentation coefficient is increased from 43 S to 48 S.

74

INITIATION OF PROTEIN SYNTHESIS

[6]

sociating with polyribosomal mRNA are easily resolved in the mono- and polysomal region of the gradient (Fig. 2A). With increased centrifugation times, resolution of 60 S ribosomal subunits from 66 S small ribosomal subunit dimers, and inactive 75 S 40 S:60 S ribosomal subunit couples from active 80 S monosomes is achieved (Fig. 2C).

Met-tRNA s and Initiation Factor Binding Systems Using Highly Purified Translational Components. In highly fractionated systems, the direct binding of ['~H]Met-tRNAf to 40 S and 43 S preinitiation complexes is highly sensitive to the energy charge of the guanine nucleotide pool. Complex stability and the apparent efficiency of translational component utilization is greatly increased by exchange of bound GTP for the nonhydrolyzable GTP analog GDPNP, or by incorporation of a GTP-regenerating system throughout the sucrose gradient. This is illustrated in Fig. 3. Incubation of 45 pmoi each of [14C]eIF-2, [3H]MettRNAf, and 40 S ribosomal subunits without taking these precautions results in the binding of 4 pmo] each of eIF-2 and Met-tRNAf in the 40 S region of the gradient. However, a lack of complex stability during centrifugation is indicated by the extensive "trail" of released eIF-2 and methionine (Fig. 3A). The presence ofa PEP-pyruvate kinase regenerating system in the sucrose gradient permits the isolation of a distinct, stable 40 S preinitiation complex containing 10 pmol each of elF-2 and Met-tRNAf (Fig. 3B). Identical results are obtained by adding GDPNP to the sample just prior to loading. GTP-regeneratingsystems may act by preventing GDP-mediated inhibition of Met-tRNAf binding. GDPNP, on the other hand, appears to prevent a functionally related destabilization of the 40 S and 43 S preinitiation complexes by eIF-5 when 60 S ribosomal subunit joining is prevented? ,Is Addition of e IF-5 to the 43 S [40 S. Met-tRNAf-eIF-2.eIF-3:GTP] preinitiation complex in the absence of 60 S ribosomal subunits results in a loss of bound Met-tRNAf, eIF-2, and eIF-3. When 60 S ribosomal subunits are available, Met-tRNAf is transferred to functional 80 S initiation complexes and eIF-2 and eIF-3 are released (Fig. 4A and B). The substitution of GDPNP for GTP in the reaction cocktail has no effect on 43 S preinitiation complex formation when compared to levels obtained in the absence of 60 S subunits, but prevents both the eIF-5 mediated factor release and 60 S ribosomal subunit joining (Fig. 4C and D). 1~W. C. Merrick,D. Peterson,B. Safer,M. Lloyd,and W. M. Kemper,FEBS Lett., in press, (1978).

[6]

ASSEMBLY OF THE EUKARYOTIC INITIATION COMPLEX 30 ]

i

i

I

f

75

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FIG. 3. Increased preinitiation complex stability is achieved by GTP regenerating systems or GDPNP. [:~H]Met-tRNAf and ['4C]eIF-2 binding to 40 S ribosomal subunits was studied in standard sucrose gradients (A) and in gradients containing 4 mM phosphoenolpyruvate-0. I IU of pyruvate kinase per milliliter (B). Thirty pmol each of [:~H]Met-tRNAf (8250 cpm/ pmol), [14C]elF-2 (455 cpm/pmol), and 40 S ribosomal subunits were used per 100/xl of binding cocktail. Gradients were centrifuged for 105 rain at 59,000 rpm in a Beckman SW 60 Ti rotor. Identical stabilization of all 40 S preinitiation complex components is obtained by addition of GDPNP to the reaction mixture (final concentration 10/xM)just before centrifugation.

PROCEDURE. After incubation and centrifugation as previously described, '4C-labeled initiation factor and [3H]Met-tRNAf binding can be analyzed by directly counting aqueous gradient fractions. We routinely use Hydromix containing 6% HzO for 0.2-ml gradient fractions. Samples are counted in a Packard liquid scintillation spectrometer using standard double-label procedures. Alternatively, to examine for possible deacylation, gradient aliquots may be analyzed by cetyltrimethylammonium bromide (CTAB) or cold trichloroacetic acid (TCA) precipitation. In the latter technique, an equal volume of ice-cold 10% TCA is added to samples; after several minutes, samples are plated on nitrocellulose or glass fiber filters. Both aminoacyl-tRNA and protein, but not free amino acid, are precipitated by this procedure. CTAB, on the other hand, primarily precipitates only RNA species. Unfractionated Reticulocyte Lysate. Met-tRNAf binding studies in reticulocyte lysate are more complex. While precharged Met-tRNAr can be used, its utilization and/or exchange of radiolabeled methionine with the endogenous amino acid pool is extremely rapid, so that in most cases it is more convenient to utilize free [3'~'S]methionine. This has the disadvantage

76

INITIATION OF PROTEIN SYNTHESIS

[6]

[14C]elF-3

[14C]elF-2

SEDIMENTATION --

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Flo. 4. Energetics of 80 S initiation complex formation. Nonhydrolyzable GTP analogs prevent initiation factor release and 60 S ribosomal subunitjoining. Binding conditions similar to those described for Fig. 3 were used for the first 15 min of incubation; 30 pmol of 50 S ribosomal subunits; 0.5/~g of elF-4C, 0.5 ~g of elF-5, and 10 k~M GDPCP (where indicated) were added and incubation was continued for 15 min. In the presence of GTP, both [~4CleIF-2 (455 cpm/pmol) and [14C]eIF-3 ( 1930 cpm/pmol) are released prior to [3H]Met-tRNAf (8250 cpm/pmol) transfer to the 80 S initiation complex. The presence of GDPCP prevents both factor release and transfer of Met-tRNAf.

that only p~S]methionine is being directly measured, which can include both free and bound initiator and internal donor species of methionineaccepting tRNA, methionyl-tRNA synthetase complexes, peptidyl-tRNA, and completed globin chains (Fig. 2A). However, since both the a- and fl-globin chains in rabbit reticulocyte lysate contain only one stable methionine residue per chain, Met-tRNAf may be easily distinguished from methionine in peptidyl-tRNA by lability of the former in the hot TCA. For example, in Fig. 2A and B, [3~S]methionine binding to the small 40 S subunit in the absence or in the presence of edeine, is entirely labilized by hot TCA precipitation, indicating binding ofa methionyl-tRNA species. In general, CTAB precipitation of gradient fractions is the procedure of choice for determination of bound Met-tRNAf since only RNA species are precipitated. Nascent peptidyl-tRNA species in polysomes will also be detected. This greatly reduces the background of radioactivity incorpo-

[6]

A S S E M B L Y OF T H E E U K A R Y O T I C I N I T I A T I O N C O M P L E X

77

rated into released globin chains which remain near the top of the sucrose gradients following centrifugation. Trailing of hemoglobin through gradient fractions and the resulting high background counts resulting from upward displacement through the flow cell can be eliminated by unloading fl'om the bottom, but this usually results in poor gradient resolution. Direct cold TCA or Millipore analyses can be used if experimental conditions inhibit completion and release of globin chains. The use of ~4C-labeled initiation factors in the unfl'actionated reticulocyte lysate permits the study of initiation factor function and distribution in an extremely active translational system which still retains many of the physiological controls involved in translational regulation. Although normal utilization of labeled factors in reconstituted assay systems provides a certain degree of assurance that functional characteristics have not been altered by labeling, only in the unfractionated lysate can normal catalytic func'tion be examined. The criteria by which to measure unaltered function, however, are difficult to establish. The rate of translational activity in the normal reticulocyte lysate is not affected by the addition of any single initiation factor, elongation factor, tRNA species, or globin mRNA. The effect of several initiation factors can be observed under special circumstances, however. For example, addition of normal or radiolabeled elF-2 to the inhibited hemin-deficient lysate will cause a temporary restoration of translational activity, but only a stoichiometric synthesis of globin is observed. "~ The addition of eIF-3 to fluoride-oredeine-inhibited lysate will also promote dissociation of nonfunctional 80 S ribosomal couples, but again catalytic function is not observed. Normal functioning of exogenous factors in the lysate can only be assessed by determination of normal utilization within the total factor pool. It is important, therefore, to examine radiolabeled factor distribution at several different input levels to check for an approach of bound factor to the specific activity of the labeled factor (Fig. 7). This not only allows accurate determination,of the endogenous factor pool size, but checks against nonspecific binding by radiolabeled material within the factor preparation. While it is simpler in most circumstances to perform duplicate experiments to correlate Met-tRNArand initiation factor binding, this may not be advisable in lysate when it is necessary to ensure that catalytic utilization of both endogenous and radiolabeled exogenous initiation factors is occurring. Since the specific activities of most factor preparations are in the range of 500-4000 dpm/pmol, pS]Met-tRNAf binding may be measured in the presence of identical amounts of radiolabeled factor, to obtain identical experimental conditions. PROCEDURE. Gradient fractions (0.1-0.5 ml) are collected as previously described. For [aaS]methionine distribution ! ml of 0.25 M sodium acetate,

78

INITIATION OF PROTEIN SYNTHESIS

[6]

pH 5.1 containing 2% cetyltrimethylammonium bromide (CTAB) is added first, followed by 1 ml of 0.25 M sodium acetate, pH 5.1, containing 500/~g of unfractionated yeast carrier RNA. Samples are vortexed and filtered on Whatman GF/C glass fiber filters. Filtered samples are washed immediately with a 1:100 dilution of the 0.5 M sodium acetate buffer containing crude yeast RNA. Radiolabeled initiation factor distribution in reticulocyte lysate is examined at 3 or 4 different input levels over the range 1-20 pmol/ml. Approximately 100-400/A of lysate are required. It is important to examine overall changes in the rate of translation and to normalize data if the pool sizes of complexes being examined are altered. Initiation factor binding may be normalized by independently measuring another component whose presence in the complex is directly related to the factor being examined (e.g., Met-tRNAf in the case of eIF-2) or by comparing the relative intensities of Coomassie Blue staining of unique factor and ribosomal polypeptide bands following PAGE analysis. While it is not necessary to have homogeneous preparations o f radiolabeled factors, accurate estimation of the factor's specific activity is required for determination of the endogenous pool size. If specific inhibitors are used to increase pool sizes, preincubation of the iysate containing the radiolabeled factor for several minutes should be allowed to ensure isotopic equilibration. D e t e r m i n a t i o n o f the Initiation F a c t o r Pool Size

Data are initially plotted as normalized counts per unit examined; [3~S]Met-tRNA~ or [13q]globin mRNA may be convenient. Addition of sufficient radiolabeled factor should allow detection of an asymptotic approach to the specific activity of the labeled factor. The endogenous pool size of initiation factor is determined by the principle of isotope dilution according to the equation: X = (ab -

b)/c

where X -- endogenous pool size; a = specific activity of exogenous factor; b = amount of exogenous factor; and c -- specific activity of total (endogenous + exogenous pool). While distribution of radioactivity is identical to factor distribution when using homogeneous preparations, autoradiography of gradient fractions subjected to polyacrylamide gel electrophoresis is generally required to rule out binding of minor contaminants, or in the case of multisubunit factors, to ensure binding of all components. Data for the estimation of the endogenous pool size of eIF-2 are presented for the purpose of illustrating initiation factor distribution, pool size

[6]

ASSEMBLY OF THE EUKARYOTIC I N I T I A T I O N C O M P L E X

79

determination, and the use of translational inhibitors to increase pool sizes (Fig. 5). When initiation is inhibited by the peptide antibiotic edeine, a 48 S preinitiation complex containing equivalent amounts of Met-tRNAf and elF-2 accumulates in reticulocyte lysate (Fig. 2B and D). Since the level of Met-tRNAf binding to the small ribosomal subunit is increased up to 10-fold, and the addition of exogenous elF-2 has no effect on the extent of 40

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FIG. 5. Determination of the endogenous elF-2 pool size in reticulocyte lysate. Identical 200-/~1 aliquots are incubated with 0, 3, 6, 9, 12, and 15/~1 of ['4C]elF-2 (900 cpm/pmol, 0.45 /~g//A) containing either unlabeled methionine or [z~'%]methionine (final specific activity = 10+ dpm/pmol) for 5 rain at 30 °. Edeine (1 /zM) is then added, and samples are incubated for an additional 4 min. After sucrose density gradient centrifugation in a Beckman SW 41 rotor at 40,000 rpm for 4.5 hr, gradient fractions are analyzed for [3~S]Met-tRNAf and ['4C]elF-2 distribution as described. The distribution of ['4C]elF-2 at each input level is presented: 1.4/~g • 0 : 2.7, O . . . . O; 4.1, [] . . . . U]; 5.4, • • ; and 6.8, A---A. Binding of ['~S]MettRNAf to the 48 S preinitiation complex was constant at all input levels of ['4C]elF-2, but [ ~4C]elF-2 binding approached a saturation value of 2200 cpm (inset). A value of 16-20 pmol of elF-2 per milliliter of reticulocyte lysate is obtained.

80

INITIATION OF PROTEIN SYNTHESIS

[6]

binding, the endogenous pool size of elF-2 may be easily determined. [14C]elF-2 (specific activity = 900 dpm/pmol), 1.4-6.8/zg, is preincubated with 200-/zl aliquots of reticulocyte lysate for 5 rain to ensure isotopic equilibration. No effect on the rate of protein synthesis is observed. Edeine is then added (final concentration = 1 tzM), and incubation is continued for 4 min longer. [3~S]Methionine (specific activity = 900,000 dpm/pmol) is used to monitor Met-tRNAy binding in duplicate experiments, in which the contribution of [14C]eIF-2 will be negligible. After sucrose density gradient centrifugation and fractionation, [14C]eIF-2 distribution is measured. In Fig. 5, data from 5 different input levels of radiolabeled eIF-2 are shown. Note that in agreement with Met-tRNAf binding data (Fig. 2D), eIF-2 is present in both the 48 S complex (fractions 15-17) and the 66 S dimer (fractions 20 and 21). The amount of [14C]eIF-2 present in the 48 S complex for each input level of the radiolabeled factor is summed and plotted (Fig. 5, inset). Binding of [14C]eIF-2 appears to approach a limiting value of 2200 dpm when 6/zg of labeled factor has been added, suggesting that the endogenous pool size is small in comparison. Each data point is then used to calculate the endogenous pool size, and results are plotted as shown (Fig. 5, inset), so that possible sources of error can be evaluated. In this experiment, for example, low input levels of radiolabeled factor lead to an overestimation of the endogenous pool size. A major reason for this seems to be related to an extremely high affinity ofeIF-2 to glass, which results in proportionally larger losses during analysis at low input levels. As can be seen from the relationships in the preceding formula, this would result in an overestimation of eIF-2. More accurate estimations approaching a value of 4-5 pmol per 200 tzl of lysate result from the use of larger amounts of factor.

Distribution of lnitiation Factors in Lysate by Laemmli PAGE Analysis Sample Preparation. The major problem encountered in Laemmli PAGE analysis of sucrose gradient fractions is the quantitative recovery of protein from very dilute solutions. In addition, conditions required for the optimal resolution oflysate components must be balanced with the amount of material required for PAGE analysis. In reconstituted systems the latter does not usually pose any problems, since the concentration of highly purified components is determined by experimental conditions. PAGE analysis of the unfractionated reticulocyte lysate, on the other hand, is more complex. Greater than 98% of the protein is globin, which hinders analysis of fractions near the top of the gradient. In addition, the pool sizes of ribosomal subunits are usually quite small. The use of specific inhibitors to increase the size of various pools being examined can be advantageous.

[6]

A S S E M B L Y OF T H E E U K A R Y O T I C I N I T I A T I O N C O M P L E X

81

Approximately 0.1-0.3 A ~s0unit of each ribosomal subunit are required for adequate visualization of ribosomal proteins. The amount required for direct visualization of bound components will vary with the relative homogeneity of the pool or the stability of component binding. PROCEDURE. Exactly 2 volumes of acetone are added to gradient fractions. After thorough mixing, the samples are allowed to precipitate for i hr at --20 ° . Above sucrose concentrations of 35% or when larger amounts of acetone are used, gradient fractions may become cloudy, indicating coprecipitation of sucrose. This can be readily reversed by the addition of water (just sufficient to restore clarity) without significant loss of precipitated protein. Previously, 10% TCA was used initially to precipitate protein and to remove sucrose. This procedure is complicated, however, by the requirement that all traces of TCA be removed by multiple acetone extractions. Loss of protein during this multistep procedure is difficult to control. Since recoveries of radiolabeled initiation factors tested were greater than 95% by acetone precipitation, this simple one-step procedure was chosen. For PAGE analysis in 0.75 mm slab gels, a sample volume no greater than 25 pJ is required. It is essential, therefore, to collect the acetone precipitate in conical centrifuge tubes and to thoroughly remove the supernatant. For gradient fractions of 0.5 ml or less, several tabletop centrifuge systems utilizing convenient 1.5 ml polyethylene tubes are available. After centrifugation (approximately 8000 g for 10 min), the supernatant is immediately decanted, tubes are inverted, and acetone is allowed to drain. Traces of remaining acetone are removed by gentle heating at 37°. Sample buffer is then immediately added, and incubation with occasional vortexing is performed at either 37° for 30 min or 90° for 5 min. The sample is then collected by centrifugation at room temperature and immediately analyzed by PAGE exactly as described by Anderson et al. TM One problem encountered using this procedure is the distortion of polypeptide bands in overloaded channels. This appears to result from physical distortion of the stacking gel geometry by ribosomal RNA and may be eliminated by decreasing the amount of sample loaded. PAGE analysis of gradient fractions is absolutely required when using partially purified initiation factor preparations to ascertain that bound radioactivity is the desired factor rather than contaminants in the preparation. In addition, the subunit composition of factors before and after binding should be checked. Following electrophoresis, gels are fixed and stained in 0.25% Coomassie Blue-50% methanol-7.5% acetic acid for 30 rain, then rapidly destained by several changes of the previous solution ~' C. W. A n d e r s o n , P. R. Baum, and R. F. Gesteland, J. Virol. 12, 241 (1973).

82

INITIATION OF PROTEIN SYNTHESIS

[6]

without stain. Gels are photographed and dried, and autoradiography is performed using Kodak type X-Omat film. Exposure times range from 3 to 20 days using 14C-labeled initiation factors. In Fig. 6, binding data for radiolabeled eIF-2, eIF-3, and Met-tRNAf during 40 S preinitiation complex formation are shown to illustrate these analytical techniques when using highly purified components. Identification of Preinitiation Complex Components in Lysate. By definition, initiation factors are those polypeptide components that promote the sequential assembly of Met-tRNAf, mRNA, and ribosomal subunits during formation of the 80 S initiation complex. Although 9 factors have been isolated that stimulate peptide bond formation with Met-tRNAy when 0.5 M KCl-washed ribosomal subunits are used, the sequence of their function, binding, and release during the initiation process is still uncertain, and it is not certain whether all participating components have been identified. It is extremely important, therefore, to isolate, identify, and characterize all intermediary complexes formed during initiation. One basic strategy is to use inhibitors that block the initiation sequence at specific points, but also allow normal elongation and termination so that ribosomal subunits needed for the accumulation of complexes above the block become available. If specific complexes can be isolated, polypeptide components can be characterized with respect to known initiation factors (by molecular weight or exact coelectrophoresis if known purified factors are available) and the functional dependence of their binding. For example, the inhibitor edeine blocks 60 S ribosomal subunit joining. 2° If mRNA is available, a 48 S complex containing equivalent amounts of Met-tRNAf and mRNA accumulates in the reticulocyte lysate. Fractionation by sucrose density gradient centrifugation and PAGE analysis show the presence of at least 8 polypeptide bands not present in the remaining 43 S complex (Fig. 8). Using purified standards, six of these can be identified as factors and/or factor subunits eIF-2, eIF-3, eIF-4A, eIF-4B, and possibly eIF-5. It is important to rule out artifactual associations of proteins with ribosomal subunits. One method is to show that such binding is functionally dependent on another preinitiation complex component, such as Met-tRNAf or mRNA. Another is to isolate the component and demonstrate activity in specific assay systems. This approach is being utilized on the two indicated, but unidentified, components in Fig. 7. The determination of the initiation factor composition of intermediary complexes formed during initiation coupled with the use of other specific inhibitors or physiological states which effect specific translational control mechanisms should offer a powerful approach toward understanding eukaryotic translation. 2oT. Obrig, J. Irvin, W. Culp, and B. Hardesty, Eur. J. Biochern. 21, 31 (1971).

[14C]

elF-2 24

SEDIMENTATION

[14C] elF-3 SEDIMENTATION

24 1E 16 I:E

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12 pmol (17%) OF Met-tRNAf AND 12 pmol (32%) OF elF-2 ARE BOUND FIG. 6. Polyacrylamide gel analysis of [14C]elF-2 and [14C]elF-3 binding to 40 S ribosomal subunits. Five micrograms of [14C]eIF-2, 14/xg of unlabeled elF-2 or 14/xg of [14C]elF-3, and 5 txg of unlabeled elF-2 were incubated with 30 pmol of 40 S ribosomal subunits and 80 pmol of purified [14C]Met-tRNAt as described. After sucrose density-gradient analysis in a Beckman SW 60 rotor at 59,000 rpm for 105 min, gradient fractions were analyzed for total ~4C counts ([14C]Met-tRNAf plus radiolabeled factors) and hot trichloroacetic acid precipitable radioactivity. An aliquot was also prepared for Laemmli sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both the autoradiograms and stained slab gels are shown corresponding to the distribution of [14C]eIF-2 and [J4C]elF-3. The specific activities of [~4C]Met-tRNAf, [~4C]elF-3, and [~4C]eIF-2 were 10,000 cpm//zg, 2800 cpm//zg, and 10,000 cpm//xg, respectively.

84

[6]

INITIATION OF PROTEIN SYNTHESIS FRACTION NUMBER

4

8

12

16

20

1.0

0.5

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~-.- ~

70,000 - 50,000

20,000

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-

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iz

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L A E M M L I SDS PAGE A N A L Y S I S

FIG. 7. Initiation factor composition of the 48 S preinitiation complex. One milliliter of reticulocyte lysate was incubated in the presence of 1 /.tM edeine as described in Fig. 5, centrifuged on a 15-35% linear sucrose gradient at 24,000 rpm for 11 hr in a Beckman SW 27 rotor at 2°. The gradient was fractionated into 1-ml aliquots, which were prepared for polyacrylamide gel electrophoresis in 75-mm 15% acrylamide slab gels as described. The Coomassie Blue-stained polypeptide patterns obtained are aligned beneath their corresponding gradient fractions. The following initiation factors were identified by exact coelectrophoresis of highly purified or homogeneous initiation factors: elF-2, II; elF-3, • (elF-4A, O, and elF-4B, O, may also be present). Other polypeptide bands associated with the 48 S complex, indicated by [] and A, but not seen in 43 S preinitiation complexes, may be elF-5 and mRNP proteins; these are currently being evaluated. The approximate relationships between the molecular weights (My) of proteins and the observed R r is this gel system are indicated.

[6]

ASSEMBLYOF THE EUKARYOTIC INITIATION COMPLEX

85

M e s s e n g e r R N A Distribution in the R e t i c u l o c y t e L y s a t e as D e t e c t e d by [5'-'~H] P o l y u r i d y l a t e H y b r i d i z a t i o n

Determination of m R N A distribution in reticulocyte lysate can provide an important approach to the problem of how translational control is mediated at the level of m R N A binding. The use of exogenous initiation factors, m R N A (especially in the RNase-treated lysate2~), and inhibitors can be used to alter rates of protein synthesis. Analysis of [5'-:~H] polyuridylate hybridization to m R N A in sucrose gradient fi'actions of reticulocyte lysate is relatively simple. The size of the globin m R N P is well established ' ' - ' 5 and the length of the polyadenylate tract has been determined. "~-'-'~Many procedures for estimating m R N A content by [3H] polyuridylate hybridization have been described, '-''~-3j including procedures for investigating m R N A localization in fractionated cell lysates. 3' However, stringent conditions must be established if the method is to be useful quantitatively as well as qualitatively in the determination of m R N A localization. Favre et al. :~3have reported that polyadenylate tracts present in m R N A protein particles have a behavior distinct from that of synthetic polyadenylate or polyadenylate tracts in m R N A when titrated with polyuridylate. Binding ofpolyuridylate is influenced by the proteins bound to the polyadenylate tract. Addition of sodium dodecyl sulfate (SDS, final concentration 0. I%) to gradient fractions before hybridization increases [3H]polyuridylate binding from 10 ~ 30%. The extent of the increase varies with the fraction examined; SDS has the greatest effect on ['~H] polyuridylate binding at the top of the gradient (15 S ~ 30 S) and the least effect in the polysomal region. PROCEDURE. Each gradient fraction is made 0.1% with respect to SDS. Hybridization is carried out in the presence of 3 × SSC (0.45 M NaC1, 4.5 m M sodium citrate, 15 m M Tris, pH 7.2) for 30 rain at 40 °. If 100 p~l of 21 H. R. B. Pelham and R. J. Jackson, Eur. J. Biochem. 67, 247 (1976). z2 A. Burny, G. Huez, G. Marbaix, and H. Chantrenne, Bioehim. Biophys. Acta 190, 228 (1969). ~3 B. Lebleu, G. Marbaix, G. Huez, J. Temmerman, A. Burny, and H. Chantrenne, Eur. J. Biochem. 19, 264 (1971). 24C. Morel, B. Kayibanda, and K. Scherrer, FEBS Lett. 18, 84 (1971). 25G. Huez, A. Burny, G. Marbaix, and B. Lebleu, Biochim. Biophys. Acta 145, 629 (1967). 2, L. Lira and E. S. Canellakis, Nature (London) 227, 710 (1970). 27j. A. Hunt, Biochem. J. 131,327 (1973). ~s A. E. Sippel, J. G. Stavrianopoulos, G. Schultz, and P. Feigelson, Proc. Natl. Acad. Sei. U.S.A. 71, 4635 (1974). 2, D. Gillespie, S. Marshall, and R. P. Gallo, Nature (London), New Biol. 236, 227 (1972). 3oN. Sullivan and W. K. Roberts, Biochemistry 12, 2395 (1973). 3J j. O. Bishop, M. Rosbash, and D. Evans, J. Mol. Biol. 85, 75 (1974). 32M. Rosbash and P. J. Ford, J. Mol. Biol. 85, 87 (1974). .~3A. Favre, C. Morel, and K. Scherrer, Eur. J. Biochem. 57, 147 (1975).

86

[6]

INITIATION OF PROTEIN SYNTHESIS

reticulocyte lysate are analyzed on a gradient and fractionated into 40 × 0.4-ml fractions, 0.03/~g of [5'-3H]polyuridylate is added to each (specific activity = 1330 X 103 cpm/~g). Unhybridized [3H]polyuridylate is digested with ribonuclease A (20 ~g/ml) at 30° for 1 hr. The ribonucleaseresistant hybrids are precipitated with 1 ml of a 3% CTAB solution in 3 mM sodium acetate buffer, pH 5.2; 1 ml of a yeast RNA solution (1 mg/ml in 0.5 M sodium acetate, pH 5.2) is added for maximum precipitation. After precipitation for 5 min, the samples are filtered through Whatman GF/C glass fiber filters and rinsed immediately with a 1/1000 dilution of the sodium acetate buffer containing RNA. The precipitate on the dried filters is digested with 0.2 ml of NCS at 40° for 1 hr. Radioactivity is determined in LSC scintillation cocktail containing 1 ml of glacial acetic acid per liter of cocktail. With each set of reactions, polyadenylate standards of known concentrations are included. Ribonuclease-resistant hybrids are found to be triplexes; 1000 cpm are found to be equivalent to 0.0345 pmol of globin mRNA (assuming an average polyadenylate tract length of 43 nucleotides). Also included are various controls: (1) precipitation of a known quantity of

0.9

II

0.6

_z~ 03

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0

i i

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I

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FRACTION NUMBER

FI~. 8. Globin mRNA and Met-tRNAt distribution in the edeine-inhibited reticulocyte lysate. Duplicate 100-/zl reaction cocktails containing nonradioactive or [35S]methionine (105 dpm/pmol) were incubated and analyzed as described for Fig. 5. Globin mRNA and [:~5S] methionine distribution were analyzed as described. An equivalent amount ofglobin mRNA and Met-tRNAf is found in the 48 S preinitiation complex. Note that the 43 S complex no longer binds Met-tRNAe and that nonribosomal bound globin mRNA has an approximate sedimentation coefficient of 30 S.

[7]

E U K A R Y O T I C I N I T I A T I O N FACTORS

87

[3H]polyuridylate, (2) precipitation of the same quantity of [3H] polyuridylate after ribonuclease digestion; (3) precipitation of [3H] polyuridylate after 60 min of incubation at 30° will all the standard solutions used in the assay, to screen for possible ribonuclease contamination. It is possible that the polyadenylate tract length on mRNA may vary in different lysate preparations, since the degree of maturity of the reticulocytes obtained may vary. If polyadenylate tract lengths are considered to be a function of age of the mRNA, populations of mRNA molecules with different average polyadenylate tract lengths might be expected from cells at different stages of maturity. An example of [3H]polyuridylate hybridization to sucrose gradient fractions is shown in Fig. 8. The example chosen is the analysis ofa reticulocyte lysate incubated in the presence of edeine (experimental conditions as described in figure legend). It shows mRNA localization in the 48 S complex and its dimer and to a lesser extent in the 80 S region. It can be seen that equivalent amounts of mRNA and Met-tRNAf are found in the 48 S complex. Globin mRNA is also found consistently in a complex near the top of the gradient, which]s found to be approximately 30 S. It is not known what relationship this complex has to the 15 S and 20 S mRNPs described by Vincent et al. 34 Further investigations of this complex are in progress. Acknowledgments We would like to thank Dr. W. French Anderson for e n c o u r a g e m e n t , advice, and generous support, and Mrs. E x a Murray for editorial assistance. .~4 A. Vincent, O. Civelli, J. Buri, and K. Scherrer. FEBS Lett. 77, 281 (1977).

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Purification of Four Eukaryotic Initiation Factors Required for Natural rnRNA Translation B y H. H. M. DAHL and G. E. BLAIR

The initiation of translation in eukaryotic cells requires ribosomal subunits, initiator tRNA (Met-tRNAv) messenger RNA, ATP, GTP and as many as seven initiation factors. 1These factors are required for binding of Met-tRNAv and mRNA to the 40 S ribosomal subunit, and for joining the 40 S.mRNA.Met-tRNAv complex to the 60 S subunit, thus forming a functional initiation complex. There may be initiation factors that can M. H. Schreier, B. Erni, and T. Staehelin, J. Mol. Biol. 116, 727 (1977).

METHODS 1N ENZYMOLOGY, VOL. LX

Copyright © 1979by AcademicPress. Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4