THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Val. 262, No. 8, Issue of March 15, pp. 3826-3832,1987 Printed in U.S.A.

The ATP-dependent Interaction of Eukaryotic Initiation Factors with mRNA*

(Received for publication, August 19, 1986)

Richard D. AbramsonS, Thomas E. DeverS, T. Glen Lawson§, Bimal K. Ray§, Robert E. Thache), and William C. MerrickS From the $Department of Biochemistry, School of Medicine, Case Western Reserue Uniuersity, Cleueland, Ohio 44106 and the §Department of Biology, Washington Uniuersity, St. Louis, Missouri 63130

The interaction of three protein synthesis initiation factors, eukaryotic initiation factor (eIF)-4A, -4B, and -4F, with mRNA has been examined. Three assays specifically designed to evaluate this interaction are RNA-dependent ATP hydrolysis, retention of mRNAs on nitrocellulose filters, and cross-linking to perio- date-oxidized mRNAs. The ATPase activity of eIF-4A is only activated by RNA which is lacking in secondary structure, and the minimal size of an oligonucleotide capable of effecting an optimal activation is 12-18 bases. In the presence of ATP, eIF-4A is capable of binding mRNA. Consistent with the ATPase activity, this binding shows a definite preference for single- stranded RNA. In the absence of ATP, eIF-4F is the only factor to bind capped mRNAs, and this binding, unlike that of eIF-4A, is sensitive to m’GDP inhibition. The activities of both eIF-4A and eIF-4F are stimulated by eIF-4B, which seems to have no specific independ- ent activity in our assays. Evidence from the cross- linking studies indicates that in the absence of ATP, only the 24,000-dalton polypeptide of eIF-4F binds to the 5’ cap region of the mRNA. From the data pre- sented in conjunction with the current literature, a suggested sequence of factor binding to mRNA is: eIF- 4F is the first initiation factor to bind mRNA in an ATP-independent fashion; eIF-4B then binds to eIF- 4F, if in fact it was not already bound prior to mRNA binding; and finally, eIF-4A binds to the eIF-4F.eIF- 4B. mRNA complex and functions in an ATP-depend- ent manner to allow unwinding of the mRNA.

Initiation of protein synthesis is a very complex process requiring more than a dozen protein factors made up of over 30 polypeptide chains. One of the key steps in the regulation of protein synthesis is mRNA binding to ribosomes (1-15). Three eukaryotic initiation factors are specifically required for the binding of mRNA to ribosomes and these are eIF1-4A, eIF-4B, and eIF-4F (also referred to as cap binding protein I1 or cap binding protein complex); eIF-4A is functional as a single polypeptide of 46,000 daltons, eIF-4B appears to be a dimer of identical 80,000-dalton peptide chains, and eIF-4F

* This work was supported in part by United States Public Health Service Grants GM 26796 and AI 20484. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: eIF, eukaryotic initiation factor; m7G, 7-methylguanosine; Hepes, 4-(2-hydroxyethyl)-l-piperazineethane- sulfonic acid K.,,, activation constant; FSBA, 5’-pfluorosulfonylben- zoyl adenosine.

is composed of three subunits (24,000, 46,000, and 220,000 daltons), one of which is similar to eIF-4A in molecular weight and isoelectric point ((16) for reviews, see Refs. 17 and 18). In addition, ATP is also required for the binding of mRNA to ribosomes (19-24).

In order to determine the role of these proteins in the protein synthetic process, we have used three assays designed to evaluate their function. These assays are: 1) RNA-depend- ent ATP hydrolysis; 2) protein-dependent retention of mRNAs on nitrocellulose filters; 3) protein cross-linking to periodate-oxidized mRNA. While the assays are artificial compared to complete protein synthesis, they do allow for the characterization of individual components and mixtures of components. The three assays we have chosen are all depend- ent on the interaction of an initiation factor (or a combination of factors) with mRNA under ionic conditions compatible with protein synthesis and display characteristics of the over- all process (such as inhibition by the cap analog m7GDP and a requirement for ATP for maximal reaction). As the results from the assays will reflect the composition of factors, we have chosen to use levels of the factors which seem to saturate an in vitro translation assay where the overall rate of synthesis is limited by available ribosomes. The ratio of factors used is in fact close to the ratio of factors present in the reticulocyte based upon purification,’ although similar values have been reported using antibodies and two-dimensional gel electro- phoresis (25).

Recently, we have described an ATP-dependent binding of mRNA by eIF-4A, eIF-4B, and eIF-4F as judged by the retention of radiolabeled mRNA on nitrocellulose filters, as well as an RNA-dependent ATPase activity of the three factors (16, 26, 27). In this communication, we describe an extension of these studies, in which evidence is presented which indicates that eIF-4A is an ATP-dependent single- stranded RNA-binding protein which is sequence nonspecific, whereas eIF-4F is an ATP-independent cap binding protein. Based on our assay systems, eIF-4B seems to have no specific activity by itself; however, it is capable of stimulating either eIF-4A or eIF-4F.

EXPERIMENTAL PROCEDURES

Purification of Initiation Factors-Eukaryotic initiation factors eIF-4A, eIF-4B, and eIF-4F were purified from rabbit reticulocyte lysates according to the procedure described earlier (16, 26).

ATPase Assay-Reactions were carried out in a 20-pl final volume in test tubes (13 X 100 mm) and contained the following: 15 mM Hepes-KOH (pH 7.5), 80 mM KCl, 2.5 mM Mg(CH&O&, 1 mM dithiothreitol, 100 p~ [y-32P]ATP (New England Nuclear; spec& activity, 2000-4000 cpm/pmol), and the indicated amounts of eIF-

R. D. Abramson, T. E. Dever, J. A. Grifo, and W. C. Merrick, unpublished results.

3826

Initiation Factors Which Interact with mRNA 3827

4A, eIF-4B, eIF-4F, and RNA. The reactions were incubated at 37 "C for 15 min and were stopped by the following sequential additions: 0.5 ml of a mixture of 20 mM silicotungstate and 20 mM sulfuric acid; 1.2 ml of 1 mM KPO, (pH 7.0); 0.5 ml of 5% ammonium molybdate in 4 M sulfuric acid; 300 pl of a mixture of 5% trichloroacetic acid and 100% acetone (1:l); and finally 2 ml of a mixture of isobutyl alcohol and benzene (1:l). It was important to keep the samples at 4 "C during these additions to minimize the nonenzymatic generation of Pi from ATP. The tubes were mixed vigorously for 30 s and centrifuged in an IEC clinical centrifuge at 1500 rpm for 3 min. Under these conditions phosphate that was released from ATP was seques- tered in the organic (upper) phase. Unhydrolyzed ATP remained in the aqueous (lower) phase. Phosphate released was quantitated by measuring radioactivity contained in a 500-p1 aliquot of the organic phase using scintillation solution (Formula 963, New England Nu- clear) and scintillation spectrometry. This was corrected for total 32Pi released, and fmol of ATP hydrolyzed were quantitated.

Globin mRNA Purification and 3' End Labeling-Globin mRNA was purified from rabbit reticulocyte lysate as described in a previous communication (16). Full length 9 S globin mRNA was labeled at its 3' end using poly(A) polymerase and [3H]ATP, essentially as de- scribed by Sippel (28). A typical labeling reaction contained 50 mM Tris-HC1 (pH 8.0), 10 mM MgSO,, 2.5 mM MnCl,, 100 pg of bovine serum albumin (nuclease free; Bethesda Research Laboratories), 220 mM NaCI, 400 units of RNasin ribonuclease inhibitor (Promega Biotec), 110 pg of globin mRNA, 400 pCi of [2,8-3H]ATP (specific activity, 41 Ci/mmol; ICN Radiochemicals), and 1.46 units of poly(A) polymerase (Pharmacia P-L Biochemicals). The mixture was incu- bated at 37 "C for 15 min in a total volume of 200 pl. Radioactive mRNAs were isolated after phenol extraction followed by ethanol precipitation and further purified by sedimentation through a 5-25% linear sucrose gradient. The purified [3H]mRNA had a specific activ- ity of 100,000 cpm/pmol corresponding to the addition of approxi- mately 10 A residues/molecule. The radiolabeled mRNA was shown to be full length by 2% agarose gel electrophoresis under nondena- turing conditions (29).

Protein-dependent Retention of mRNA on Nitrocellulose Filters- The retention of 3' end-labeledglobin mRNA or 13H]poly(A) (specific activity, 6.24 pCi/pmol of nucleotide phosphorus; Miles Laboratories Inc.) on nitrocellulose was performed under conditions where mRNA is not retained on nitrocellulose unless it is bound to protein as described (26). Reactions were performed in test tubes (13 X 100 mm) and incubated at 37 "C for either 2 min (with globin mRNA) or 3 min (with poly(A)) in a total volume of 50 pl containing 20 mM Hepes- KOH (pH 7.5), 90 mM KCl, 5 mM Mg(CH3C02),, 1 mM dithiothreitol, 3 mM phosphoenolpyruvate, 0.3 IU of rabbit muscle pyruvate kinase, and the indicated amount of initiation factors and radiolabeled RNA. Where indicated, 2 mM M$+/ATP and 2 mM Mg'+/m7CDP were included. The reaction was stopped by the addition of 2 ml of ice- cold wash buffer (20 mM Tris-HC1, pH 7.5, 1 mM dithiothreitol, 0.1 mM EDTA, 2.5 mM Mg(CH,CO,),, and either 100 mM KC1 (with globin mRNA) or 20 mM KC1 (with poly(A)) and immediately applied to nitrocellulose filters. The tubes were rinsed twice with cold buffer being careful not to allow the filter to go dry until the last rinse was applied. Radioactivity was determined using scintillation solution (Formula 963, New England Nuclear) and scintillation spectrometry.

Chemical Cross-linking of Factors to mRNA-Reovirus mRNA labeled at the 5' cap with [a-32P]GTP (30) (specific activity, 2.7 X lo6 cpm/pg) was oxidized with NaIO, according to Muthukrishnan et al. (31). It was then incubated with initiation factors for 10 min at 30 "c in reaction mixtures (25 pl) containing 20 mM Hepes-KOH (pH 7.5), 100 mM KCI, 5 mM Mg(CH&O&, 1 mM dithiothreitol, 3 mM phosphoenolpyruvate, and 0.3 IU of rabbit muscle pyruvate kinase. Where indicated, 2 mM M$/ATP and 2 mM M$/m7GDP were included. Reversibly cross-linked complexes formed between protein and mRNA were stabilized by reduction with NaBH3CN according to Sonenberg et al. (32). Proteins were then precipitated by addition of 500 /.d of acetone, 1 M HCl (120:l v/v), washed twice with ethanol/ether (1:l v/v), lyophilized to dryness, and dissolved in 15 p1 of a solution containing 3 M urea, 26 mM EDTA (pH 7.1), 0.52 mg of RNase A (Boehringer Mannheim)/ml, and 260 units of RNase TI (Boehringer Mannheim)/ml. The mixture was incubated for 4 h at 37 "C, and the radioactive proteins were fractionated on a 12.5% polyacrylamide gel (ratio of acrylamide to bisacrylamide = 58.6) in the presence of sodium dodecyl sulfate (33) and analyzed by autora- diography (RP X-Omat AR x-ray film, Kodak).

RESULTS

Previously we have described an RNA-dependent ATPase assay using eIF-4A, eIF-4B, and eIF-4F (27). The normal situation involves the use of globin mRNA, and as a nonspe- cific control, poly(U) was tested in the same assay. For globin mRNA, little ATP hydrolysis is observed except with eIF-4F (in the presence of eIF-4A and/or eIF-4B), and this hydrolysis is sensitive to inhibition by the cap analog, m7GDP. In con- trast, eIF-4A is quite active by itself when poly(U) is the activator, although this activity is stimulated by eIF-4B (which has no activity by itself). With poly(U), the activity of the three factors is approximately additive over the combi- nations of eIF-4A + eIF-4B and eIF-4F + eIF-4B, whereas when globin mRNA was the activator, a 3-fold synergism was noted. As might be expected, the activities with poly(U) were not sensitive to inhibition by m7GDP.

In order to further characterize this activity, we have looked at the ability of a large variety of RNAs to stimulate eIF-4A- dependent ATP hydrolysis. The results of this experiment are presented in Table I. As can be seen, ribohomopolymers lacking secondary structure are effective as activators of the eIF-4A RNA-dependent ATPase. Other RNAs (those with considerable secondary structure or double-stranded RNAs) do not serve as activators nor do single-stranded DNAs. The only ribohomopolymer which is a poor activator is poly(G), and this is presumably due to its tendency to form two- and three-stranded helices under the experimental conditions (34). Therefore, the specificity of eIF-4A appears to be for single-stranded RNAs which lack secondary structure.

In addition, kinetic studies were done on the ability of the five ribohomopolymers to activate the ATPase activity of eIF- 4A. Table I1 presents the results of these experiments. The differential ability of the ribohornopolymers to activate ATP hydrolysis is reflected in their different V,,, values, since all five seem to have a similar KaCt. The similar K,,, values suggest that eIF-4A has a similar, if not equal, ability to form a preliminary complex with RNA regardless of structure or sequence. The ability to form a productive activated complex which leads to ATP hydrolysis, however, is not equal, and this is indicated by the different V,,, values seen. The fact that poly(G) has a large amount of secondary structure and is a poor activator of the ATPase activity is reflected in its

TABLE I RNA-dependent ATP hydrolysis by eIF-4A

Reactions were carried out in the presence of 100 PM [Y-~'P]ATP, 1.9 Kg of eIF-4A, and the indicated amounts of RNA or DNA. Activity was measured by the release of 32P1 and quantitated for fmol of ATP hydrolyzed. A background of Pi released in the absence of protein and RNA of 6.9 fmol/s was subtracted from each value.

Activator AZM Pi released

unit fm0 l i g . s None 3.4 POlY(C) 0.31 POMA)

47.4 0.25 37.4

POlY(U) 0.29 POlY(1)

35.4

Poly(G) 0.30 0.31 31.6

5.7

Globin mRNA 0.28 tRNA

11.2 0.26 13.2

Poly(1-C) 0.25 6.2 Poly(A) .poly(U) 0.25 11.0

Oligo(dT) 0.30 Poly(dT)

2.6 0.27

POlY(dA) 4.6

0.37 5.8

Initiation Factors Which Interact with mRNA TABLE I1

K,, and V,, for RNA-dependent ATP hydrolysis by eIF-4A The RNA-dependent ATP hydrolysis assay was performed using

the various indicated ribohomopolymers as activators. The amount of activator was varied from approximately 0.02 to 0.90 Am unit/ assay. Kact and V,. were determined by a double reciprocal plot of 1 /V versus l/[activator]. Conversion of AZM) units to p~ concentra- tions of poly(N) was based on an average length of 100 nucleotides/ polymer and the following molar extinction coefficients at 260 nm: poly(A), 9.5 X lo3; poly(U), 9.5 X lo3; poly(C), 5.5 X lo3; poly(I), 5.8 X lo3; poly(G), 8.7 X lo3 (51).

KU-t Activator

A , Polv(N) V,.

unit W fmol Pi released M . 8

Poly(A) 0.31 16 150 POlY(U) 0.29 15 110 POlY(C) 0.23 21 70 POlY(1) 0.17 15 60 Poly(G) 0.31 18 20

TABLE I11 Influence of oligomer size on ATP hydrolysis by eiF-4A

Reactions were carried out in the presence of 100 p~ [T-~~PIATP, 2.3 pg of eIF-4A, and the indicated amounts of RNA. Activity was measured by the release of 32Pi and quantitated for fmol of ATP hydrolyzed. A background of Pi release in the absence of protein and RNA of 7.8 fmolh was subtracted from each value.

Activator A,, Pi released

unit fm0llwg.s None 9.0 Oligo(A), 0.36 13.3 Oligo(A), 0.36 31.2 Oligo(A)lz-ls 0.32 89.4 Poly(A)>sc+ 0.25 84.9

extremely low Vmax. This further suggests that only single- stranded RNAs lacking secondary structure are capable of activating the ATP hydrolysis property of eIF-4A and that this activation is independent of sequence.

The data in Table I11 represents an attempt to determine the number of nucleotides necessary for the activation of the ATP hydrolysis activity of eIF-4A. As can be seen from the table, the minimal size of an oligonucleotide capable of ef- fecting an optimal activation of eIF-4A is 12-18 bases. An oligo(A) octamer is only about 25% as effective in activating the ATPase activity, whereas an oligo(A) tetramer is virtually unable to activate eIF-4A.

As a more direct test of the preference of eIF-4A for single- stranded RNAs as activators, an experiment was performed to test the ability of eIF-4A to respond to an mRNA with “increasing secondary structure”. To achieve this RNA, poly(U) was tested for its ability to stimulate eIF-4A in the presence of increasing amounts of oligo(A)12-18 (Fig. 1). Indi- vidually, both poly(U) and oligo(A)12-ls stimulate the eIF-4A- dependent hydrolysis of ATP. However, as can be seen in Fig. 1, when both are added (under conditions which allow the oligo(A)12-18 to hybridize to the poly(U)) there is a loss of activity. This loss in activity begins when the amount of oligo(A)12-1R (as [Ap]) is about 10% the level of poly(U) (as [Up]), and essentially complete loss of activity is noted a t a ratio of [Ap] to [Up] of about 2 to 3. The fact that less than a 1 to 1 ratio of [Ap] to [Up] is sufficient to extinguish activity is interpreted as follows; at the point where 60-70% of the poly(U) is hybridized to oligo(A)ly_ls, the remaining poly(U) available (30-40%) to activate eIF-4A is represented by rela- tively short stretches of single-stranded Us (ie. on average less than poly(U)lo). As shorter oligonucleotides are less ef-

0 0.2 0.4 0.6 0.8 1.0

[%I9 mM FIG. 1. Influence of RNA secondary structure on ATP hy-

drolysis by eIF-4A. The RNA-dependent ATP hydrolysis assay was performed with the following modifications. Prior to the addition of protein and [32P]ATP, the reaction mixture was incubated at 30 “C for 10 min to allow annealing of the complementary RNAs. After the addition of protein and ATP, the reaction mixture was then incubated for an additional 15 min at 30 “C. Reactions were carried out in the presence of 100 p M [y-32P]ATP, 2.5 Kg of eIF-4A, and the indicated amounts of oligo(A)12.18 (expressed as the concentration of Ap). Where indicated 0.29 unit (1.53 mM Up) of poly(U) was included. Activity was measured by the release of 32Pi and quantitated for fmol of ATP hydrolyzed. A background of Pi released in the absence of protein and RNA of 1.1 fmol/s was subtracted from each value.

fective in stimulating the ATPase activity of eIF-4A (see Table 111), the apparent “early” loss of activity at less than 1 to 1 molar ratios of [Ap] to [Up] is thus an indication of not only a loss of available poly(U) (as double-stranded RNAs are essentially inactive, Table 1) but also a reduction in the “apparent” length of the nonhybridized poly(U) into a range where there is insufficient size to effectively interact with eIF-4A.

In a complementary series of studies, we have looked at the ability of various factor combinations to interact with globin mRNA as determined by retention of radiolabeled mRNA on nitrocellulose filters. These results are indicated in Table IV. Using globin mRNA as the substrate, eIF-4F alone functions as a single factor which can bind globin mRNA, and this binding is sensitive to m7GDP inhibition. This binding of eIF-4F is not appreciably stimulated by either eIF-4B or ATP. In contrast, eIF-4A binds globin mRNA only in the presence of ATP, and this binding is dramatically stimulated by eIF- 4B. A significant difference between this assay and the RNA- dependent ATPase assay is that there is no apparent syner- gism when all three factors are added but rather what appears to be just an additive effect. This is seen not only in total counts but also in the very modest inhibition by m7GDP.

As the control, the same experiments were performed with poly(A) as the mRNA (Table V). Since poly(A) is known to bind to nitrocellulose filters under conditions of high ionic strength, a slight modification of the protocol was needed (35). The wash buffer that was used for the transfer and washing of the reactions was changed from 100 mM KC1 (where 100% of the poly(A) bound to the filter in the absence

Initiation Factors Which Interact wi th mRNA 3829

TABLE IV PHIGlobin mRNA binding

Initiation factor binding to globin mRNA was determined by the retention of [3H]globin mRNA on a nitrocel- lulose filter after incubation with the indicated factors both in the presence and absence of 2 mM M e / A T P and/ or 2 mM Mg2+/m7GDP. Reaction mixtures containing 20,000 cpm of [3H]mRNA (0.2 pmol), 3.0 pg of eIF-4A, 1.1 pg of eIF-4B, and 1.2 pg of eIF-4F were incubated for 2 min at 37 "C. A background of 400 cpm obtained in the absence of added protein was subtracted from each value.

eIF-4A eIF-4A and eIF-4B eIF-4B eIF-4F and

eIF-4B eIF-4F eIF-4A, eIF-4B. and eIF-4F

+m7GDP

cpm bound PHlglobin mRNA 130 150 230 3,820 3,610 4,020 70 190 20 1,050 1,170 1,000

+ATP 1,800 5,170 350 4,260 3,990 10,650 +ATP + m7GDP 1,270 4,320 (-60) 1,200 1,570 8,130

TABLE V rH]Poly(A) binding

Initiation factor binding to poly(A) was determined by the retention of [3H]poly(A) on a nitrocellulose filter after incubation with the indicated factors both in the presence and absence of 2 mM M e / A T P and/or 2 mM M%+/m7GDP. Reaction mixtures containing 15,000 cpm of [3H]poly(A) (2.3 pmol), 3.0 pg of eIF-4A, 1.6 pg of eIF- 4B, and 0.75 pg of eIF-4F were incubated for 3 min at 37 "C. A background of 460 cpm obtained in the absence of motein was subtracted from each value.

eIF-4A eIF-4A and eIF-4B eIF-4B eIF-4F and

eIF-4B eIF-4F eIF-4A, eIF-4B, and eIF-4F

+m7GDP

cpm bound PHlpoly(A) 20 530 210 500 110 620 70 580 165 360 180 650

+ATP 470 5090 120 1080 120 6920 +ATP + m7GDP 470 4320 110 740 170 5600

of protein) to 20 mM KC1 (where less than 2% of the poly(A) bound to the filter in the absence of protein). As can be seen from the table, eIF-4F binds relatively little poly(A) by itself, and this binding is only slightly increased by eIF-4B. By itself, eIF-4A binds some poly(A) in an ATP-dependent manner, but this binding is dramatically increased by the presence of eIF-4B. As with globin mRNA, there appears to be no syner- gism noted when all three factors are added, but rather an additive effect. As was observed in the RNA-dependent ATP- ase assay, there appears to be no inhibition by the cap analog, m7GDP. These findings suggest that the observed inhibition by m'GDP is due to a decreased ability to bind the 5' cap of the globin mRNA and not a nonspecific interaction ( i e . not an allosteric effect or competition for the substrate nucleotide, ATP). In addition, the data suggest that the primary site of m7GDP inhibition is through eIF-4F.

A third assay, cross-linking to oxidized 32P cap-labeled reovirus mRNA, is designed to specifically investigate those factors which interact with the 5' cap structure of mRNA. Fig. 2 shows the cross-linking of the three single factors, eIF- 4A, eIF-4B, and eIF-4F, in the presence and absence of ATP and/or m7GDP. As has previously been shown (16), only the 24,000-dalton subunit of eIF-4F (also referred to indepen- dently as cap binding protein I or eIF-4E, with a molecular weight of 24,000-28,000 daltons) cross-links to the cap in the absence of ATP, and this cross-linking is extremely sensitive to m7GDP inhibition (lanes 9-12). On the other hand, eIF-4A can be shown to cross-link only in the presence of ATP (lunes 1-4); however, the extent of cross-linking is considerably less than that of the 24,000-dalton polypeptide of eIF-4F, and this cross-linking is not sensitive to m7GDP. No specific interac- tions can be seen with eIF-4B (lunes 5-8).

Fig. 3 is a repeat of the cross-linking experiment using various combinations of the factors. When both eIF-4A and eIF-4B are present (lunes 1-3), the ATP-dependent cross-

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

Mr x

- 80

- 46

- 24

ATP - - + + " + + - - + + m7GDP - + - + - + - + - + - +

FIG. 2. Cross-linking of single initiation factors to oxidized 32P cap-labeled reovirus mRNA. Oxidized reovirus mRNA labeled at the 5' cap site with [cI-~*P]GTP was incubated with initiation factors for 10 min at 30 "C; 2 mM M e / A T P and 2 mM Me/m'GDP were included as indicated. The samples were then treated with NaBH3CN for 2 h a t 4 "C followed by RNase digestion. Radioactive proteins were fractionated on a 12.5% polyacrylamide gel in the presence of sodium dodecyl sulfate and analyzed by autoradiography. Lanes 1-4, 3.4 pg of eIF-4A; lanes 5-8, 1.6 pg of eIF-4B; lanes 9-12, 1.5 pg of eIF-4F.

linking of eIF-4A is slightly enhanced, although this cross- linking is still seen to be insensitive to m7GDP inhibition. When both eIF-4F and eIF-4B are present (lunes 4-6), both the 46,000-dalton subunit of eIF-4F and eIF-4B can be seen to cross-link in an ATP-dependent m7GDP-sensitive fashion, although neither cross-links to the extent that the 24,000-

3830 Initiation Factors Which Interact with mRNA 1 2 3 4 5 6 7 8 9 1011 1213

Mr ( x 10-3)

- 80

- 46

- 24

ATP - + + - + + - + + - - + + m7GDP - - + - - + - - + - + - +

FIG. 3. Cross-linking of initiation factor mixtures to oxi- dized 32P cap-labeled reovirus mRNA. Oxidized reovirus mRNA labeled at the 5' cap with [cx-~*P]GTP was incubated with initiation factors for 10 min at 30 'C; 2 mM M$+/ATP and 2 mM Mg2+/m7GDP were included as indicated. The samples were then treated with NaBH,CN for 2 h a t 4 "C followed by RNase digestion. Radioactive proteins were fractionated on a 12.5% polyacrylamide gel in the presence of sodium dodecyl sulfate and analyzed by autoradiography. Lanes 1-3, 3.4 pg of eIF-4A + 1.6 pg of eIF-4B; lanes 4-6, 1.5 pg of eIF-4F + 1.6 pg of eIF-4B; lanes 7-9,3.4 pg of eIF-4A + 1.5 pg of eIF- 4F; lanes 10-13, 3.4 pg of eIF-4A + 1.6 pg of eIF-4B + 1.5 pg of eIF- 4F.

dalton polypeptide does. Lanes 7-9 show the cross-linking in the presence of both eIF-4A and eIF-4F. An increased ATP- dependent cross-linking to the 46,000-dalton polypeptide (eIF-4A and/or the 46,000-dalton subunit of eIF-4F) can be seen, and this cross-linking is appreciably inhibited by m7GDP, although not to the extent that the 24,000-dalton polypeptide is. When all three proteins are present (lanes IO- I3), in the absence of ATP, only the 24,000-dalton polypeptide of eIF-4F cross-links, and this cross-linking is sensitive to m7GDP inhibition. However, in the presence of ATP, not only is there extensive cross-linking to the 46,000-dalton polypeptide (eIF-4A and/or eIF-4F) and to eIF-4B, there is also a concomitant loss of cross-linking to the 24,000-dalton subunit of eIF-4F. This cross-linking can be seen to be par- tially sensitive to m7GDP inhibition.

It would appear that eIF-4F (in particular the 24,000-dalton polypeptide) is the only single factor that specifically cross- links to the 5' cap of mRNA in an ATP-independent fashion. Conversely, eIF-4A and eIF-4B only interact with the cap in an ATP-dependent manner and only show m7GDP sensitivity when eIF-4F is also present. When all three proteins are present, along with ATP, a different interaction appears to result, and the 46,000-dalton polypeptides of eIF-4F and/or eIF-4A and eIF-4B (rather than the 24,000-dalton polypeptide of eIF-4F) become the major polypeptides to cross-link to the oxidized 5' cap. Since this cross-linking is only partially sensitive to m7GDP inhibition, it may not be a specific cap interaction. Rather the reduction in cross-linking which is seen may be exclusively due to an inhibition of the interaction between eIF-4F and the cap. Thus, Pelletier and Sonenberg (36) have recently shown that using crude initiation factors in the presence of ATP and photochemical cross-linking to mRNA, only a 24,000-dalton polypeptide and an 80,000- dalton polypeptide (presumably the 24,000-dalton subunit of eIF-4F and eIF-4B) cross-link in a cap-specific fashion. This difference may be due to the different mechanism by which cross-linking to the mRNAs was achieved and/or to the difference in using crude initiation factor preparations, rather than only the purified factors.

DISCUSSION

The mechanism of binding mRNA to the 40 S ribosomal subunit is a poorly understood step in eukaryotic initiation. Since the recognition of mRNA is the site of regulation in mRNA discrimination normally (13-15), in several systems following viral infection (1-8), and during heat shock (9-12), an understanding of this step is of utmost importance. Pre- viously eIF-4A, eIF-4B, and eIF-4F had been implicated in the binding of mRNA to the 40 S ribosomal subunit. In this report we have examined the ability of these three factors to interact with mRNA and ATP. The results presented here suggest that eIF-4A is an ATP-dependent single-stranded RNA binding protein which is sequence nonspecific. The ATPase activity is a catalytic function of eIF-4A (Ref. 27 and this paper), but the turnover number under current conditions is rather small (-1 ATP hydrolyzed per eIF-4A molecule per 6 min). The interaction of eIF-4A with single-stranded regions of mRNA activates its ATPase, and this activity is greatly stimulated by eIF-4B. The minimal size of an oligo(A) capable of effecting an optimal interaction with eIF-4A is 12-18 bases. I t was previously shown that eIF-4A has the ability to melt out or unwind mRNA structure in the presence of ATP, an activity stimulated by eIF-4B (30). Thus, it seems likely that in a step prior to or in coordination with the unwinding of mRNA, eIF-4A binds to single-stranded regions of the mRNA and that it is the resultant activation of the ATPase activity in eIF-4A that allows the elimination of any neighboring regions of mRNA secondary structure.

The only factor capable of interacting with mRNA in the absence of ATP is eIF-4F, and based on the cross-linking studies, this interaction has been localized to the 24,000- dalton subunit. In reactions with natural mRNAs, eIF-4F confers the property of making the assay sensitive to inhibi- tion by m7GDP. Additionally it has been demonstrated that eIF-4F possesses an ATPase activity. This activity, like that of eIF-4A, is stimulated by eIF-4B, which appears to have no independent activity of its own in our assays. Recent evidence indicates, however, that eIF-4B may be responsible for the recycling of the 24,000-dalton subunit (and possibly other subunits as well) of eIF-4F (43). We have separated the 46,000-dalton subunit from the rest of the eIF-4F complex (30), and based on our experiments with the eIF-4F fragments have localized the RNA-dependent ATPase activity to the 46,000-dalton subunit (data not shown). Ray et al. (30) have also attributed an mRNA unwinding activity to the 46,000- dalton polypeptide of eIF-4F. In contrast to eIF-4A, the unwinding capability of eIF-4F is sensitive to m7GDP, a finding consistent with those assays reported here.

Further evidence that both eIF-4A and the 46,000-dalton subunit of eIF-4F utilize ATP has been provided by Sarkar et al. (37). In their experiments, UV irradiation of eIF-4F in the presence of [w3*P]dATP/ATP resulted in the cross-linking of this compound to the 46,000-dalton polypeptide. Similarly, dATP/ATP was also found to cross-link to eIF-4A, albeit less efficiently. Using the wheat germ system, Seal et al. (38) reported that the only protein they could identify as being inactivated with the ATP analog FSBA was eIF-4A. In the reticulocyte system, we have been able to specifically label eIF-4A as well as the isolated 46,000-dalton polypeptide of eIF-4F with [14C]FSBA (39,40).'

The above findings are consistent with the observation that the 46,000-dalton subunit of eIF-4F is chemically very similar to eIF-4A. The two polypeptides have the same molecular weight and PI (16, 41). In addition, analysis of T-labeled tryptic peptides of the two proteins showed that the majority of the labeled peptides visualized was the same, with only one

Initiation Factors Which Interact with mRNA 3831

or perhaps two notable differences observed (41). In a second study, we have analyzed CNBr fragments of the two polypep- tides reductively methylated with either [3H]- or [‘4C]form- aldehyde and again found a high degree of similarity, but not identity, between the two polypeptides (42). Thus, it seems that either these are the same gene products with slight differences in post-translation modifications or that they are products of two separate, but very similar, genes.

Currently, considerable experimentation has been focused on the involvement of mRNA structure on translational reg- ulation and mRNA discrimination. Data from Godefroy-Col- burn et al. (44) indicate that availability of the m7GDP cap structure in an mRNA is correlated with efficiency of trans- lation. Our data suggests that it is eIF-4F which first recog- nizes the cap structure and is responsible for the differential translation seen. This is consistent with the observation that eIF-4F is the major discriminatory factor in protein synthesis as determined by its ability to relieve competition between different mRNAs (11, 14, 15). Other studies have focused on the influence of increased secondary structures in the 5’ noncoding region on efficiency of mRNA translation (45, 46). In general, an increase in secondary structure (as hairpin loops) decreases translational efficiency although the direct correlation of stability of these hairpin loops (AGO) to trans- lational efficiency is poorly defined. In one study, it was shown that the increased secondary structure did not appreciably interfere with the eIF-4F interaction with the 5’ cap structure of the mRNA (as judged by photochemical cross-linking) but did show a reduced interaction with eIF-4B (36).

More recently, we have found that the presence of second- ary structure involving the first 15 bases of mRNAs adjacent to the cap reduces both translatability and competitive ability of the mRNAs (52). Secondary structure further downstream (starting from base 16), however, had little or no effect. Based on cross-linking studies, this reduction is presumed to be due to a reduction of the interaction of the 46,000-dalton subunit of eIF-4F, as well as eIF-4A and eIF-4B, and not a reduction in the ATP-independent interaction with the 24,000-dalton subunit of eIF-4F, an observation similar to that of Pelletier and Sonenberg (36). The correlation of reduced translatability with a reduced interaction of eIF-4A, eIF-4B, and/or eIF-4F with a given mRNA is consistent with our observation that all three factors are necessary for maximal mRNA-dependent ATPase activity and maximal mRNA binding.

Proteins corresponding to eIF-4A, eIF-4B, and eIF-4F have been prepared from wheat germ and have properties similar to, but not identical to, those prepared from mammalian systems. Wheat germ eIF-4A is quite similar to mammalian eIF-4A in that it is active as a single polypeptide of 48,000 molecular weight and binds ATP (36,47). However, both eIF- 4B and eIF-4F appear different. Depending on the prepara- tion, eIF-4B contains either just an 80,000-dalton peptide (like mammalian systems) or both 80,000- and 26,000-dalton peptides (48, 49). The former preparation was identified as the major mRNA binding protein, and this binding was in- dependent of ATP. It was shown, however, that eIF-4A was able to aid or stabilize eIF-4B. mRNA complex formation in an ATP-requiring reaction (48).

Wheat germ eIF-4F, as characterized by reversing the in- hibitory effects of m7GDP on protein synthesis, has been prepared in three different molecular forms, each of which contains the 24,000-dalton cap binding protein (49, 50). In each preparation there appears to be no equivalent to the 46,000-dalton peptide which may reflect species differences or purification using phosphocellulose (which has been shown to dissociate the 46,000-dalton peptide from the mammalian

eIF-4F complex (30)). The other eIF-4F peptides (220,000 or 110,000 (49); 26,000 and 75,000 (50)) may represent break- down products of the highest molecular weight subunit (220,000) or isozyme forms. Cross-linking studies with oxi- dized reovirus mRNA have shown that the 24,000-dalton (or possibly the 26,000-dalton) polypeptide interacts specifically with the 5’ cap structure of the mRNA in the absence of ATP or any additional proteins (50). When eIF-4A is added to a crude preparation of eIF-4B and eIF-4F, m7GDP-sensitive ATP-dependent cross-linking can be seen to two additional polypeptides, one of which is presumably eIF-4A (50). Thus, in some ways, the wheat germ eIF-4F resembles the mam- malian eIF-4F. Although further characterization of the wheat germ factors is necessary to determine a mechanism of mRNA binding in this system, it appears that a similar mechanism to that which we present for the reticulocyte system may also be found in wheat germ.

The observations presented here in conjunction with those previously reported have led to a working model for the binding of factors to mRNA. The first initiation factor to bind mRNA is eIF-4F. It binds in an ATP-independent man- ner, and the relative availability of the cap structure is a major determinant in the competition of mRNAs for naturally lim- iting amounts of eIF-4F. Subsequently, eIF-4B binds to eIF- 4F if in fact it was not already bound to eIF-4F prior to or during its initial binding to the mRNA.3 Finally, eIF-4A binds to the eIF-4F.eIF-4B. mRNA complex and functions in an ATP-dependent manner to allow unwinding of the mRNA and may provide for Kozak’s proposed ATP-dependent scan- ning as well (23, 24). In this sequence of events, eIF-4B is thought to play a critical role in coordinating the individual activities of eIF-4F (as a “cap” recognition element) and eIF- 4A (as an mRNA unwinding element). The amount of sec- ondary structure in the 5’ noncoding region can partially determine the competitiveness of an mRNA, presumably through an increased requirement for eIF-4A. Based upon Kozak’s two-step ATP requirement for the correct positioning of mRNA on the 40 S subunit (23, 24) and our observations of the properties of eIF-4A, eIF-4B, and eIF-4F, it is likely that multiple ATP molecules are required for successful bind- ing of mRNA in the initiation process. Only further experi- mentation with an emphasis on mRNA already bound to 40 S subunits and poised to “scan” will provide the data necessary to form a more complete working model.

Acknowledgments-We wish to thank Dr. John P. Leis for helpful discussion, Dr. Robert E. Rhoads for technology aiding in our puri- fication of eIF-4F, and Jean Kee for expert editorial assistance.

REFERENCES

1. Tahara, S. M., Morgan, M. A., and Shatkin, A. J. (1981) J. Biol. Chem. 256, 7691-7694

2. Golini, R., Thach, S. S., Birge, C. H., Safer, B., Merrick, W. C., and Thach, R. E. (1976) Proc. Natl. Acad. Sci. CJ. S. A. 73, 3040-3044

3. Rose, J. R., Trachsel, H., Leong, K., and Baltimore, D. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2732-2736

4. Trachsel, H., Sonenberg, N., Shatkin, A. J., Rose, J. K., Leong, K., Bergman, J. E., Gordon, J., and Baltimore, D. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 770-774

5. Etchison, D., Milburn, S. C., Edery, I., Sonenberg, N., and Her- shey, J. W. B. (1982) J. Biol. Chem. 257, 14806-14810

Based upon evidence achieved from the purification of initiation factors, it has been shown that eIF-4B and eIF-4F form a stable complex in solution in uitro (16). Thus, it is reasonable to assume that eIF-4B and eIF-4F may exist as a stable complex in uiuo, prior to and independent of the recognition of the m’GDP cap of mRNA by eIF-4F.

3832 Initiation Factors Which Interact with mRNA

6. Lee, K. A,, and Sonenberg, N. (1982) Proc. Natl. Acad. Sci. U. S. 30. Ray, B. K., Lawson, T. G., Kramer, J. C., Cladaras, M. H., Grifo, A. 79,3447-3451 J. A., Abramson, R. D., Merrick, W. C., and Thach, R. E. (1985)

7. Etchison, D., Hansen, J., Ehrenfeld, E., Edery, I., Sonenberg, N., J. Biol. Chem. 260, 7651-7658 Milburn, S., and Hershey, J. W. B. (1984) J. Virol. 51, 832- 31. Muthukrishnan, S., Morgan, M., Banerjee, A. K., and Shatkin, 837 A. J. (1976) Biochemistry 15, 5761-5768

8. Bernstein, H. D., Sonenberg, N., and Baltimore, D. (1985) Mol. 32. Sonenberg, N., and Shatkin, A. J. (1977) Proc. Natl. Acad. Sci. Cell. Biol. 5 , 2913-2923 U. S. A. 74,4288-4292

9. Lindquist, S. (1980) J. Mol. Biol. 137, 151-158 33. Laemmli, U. K. (1970) Nature 227, 680-685 10. Scott, M. P., and Pardue, M. L. (1981) Proc. Natl. Acad. Sci. U. 34. Felsenfeld, G., and Miles, H. T. (1967) Annu. Reu. Biochem. 36,

11. Panniers, R., Stewart, E. B., Merrick, W. C., and Henshaw, E. C. 35. Brawerman, G. (1974) Methods Enzymol. 30, 605-612

12. Duncan, R., and Hershey, J. W. B. (1984) J. Bid. Chem. 259, 3230

13. Kabat, D., and Chappell, M. R. (1977) J. Biol. Chem. 252,2684- 260,13831-13837

14. Ray, B. K., Brendler, T. G., Adya, S., Daniels-McQueen, S., Sci. U. S. A. 8 0 , 6562-6565

S. A. 78,3353-3357 407-448

(1985) J. Bid. Chem. 260,9648-9653 36. Pelletier, J., and Sonenberg, N. (1985) Mol. Cell. Biol. 5 , 3222-

11882-11889 37. Sarkar, G., Edery, I., and Sonenberg, N. (1985) J. Bid. Chem.

2690 38. Seal, S. N., Schmidt, A., and Marcus, A. (1983) Proc. Natl. Acad.

ill^^, J. K., ~ ~ ~ ~ h ~ ~ , J. w. B,, if^, J. A,, Merrick, w. c., 39. Abramson, R. D., Caliendo, A. M., Grifo, J. A., and Merrick, w . and Thach, R. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,

40. Anthony, D. D., Dever, T. E., Abramson, R. D., Lobar, M., and C. (1985) Fed. Proc. 44, 1223

Merrick, W. C. (1986) Fed. Proc. 45, 1768

S., Hershey, J . W. B., Trachsel, H., and Sonenberg, N. (1983) J. Biol. Chem. 258,11398-11403

42. Merrick, W. C., Abramson, R. D., Anthony, D. D., Jr., Dever, T. E., and Caliendo, A. M. (1987) in Translntioml Regulation of Gene Expression (Ilan, J., ed) Plenum Publishing Corp., New York, in press

43. Ray, B. K., Lawson, T. G., Abramson, R. D., Merrick, W. C., and Thach, R. E. (1986) J. Biol. Chem. 261, 11466-11470

Eur. J . Biochem. 147,549-552

663-667 15. Sarkar,G., Edery,I.,Gallo,R.,andSonenberg,N. (1984)Biochim. 41. Edery, I., Hiimbelin, M., Darveau, A,, Lew, K. A. w.,

Biophys. Acta 783, 122-129 16. Grifo, J. A,, Tahara, S. M., Morgan, M. A., Shatkin, A. J., and

Merrick, W. C. (1983) J. Biol. Chem. 258, 5804-5810 17. Moldave, K. (1985) Annu. Rev. Biochem. 5 4 , 1109-1149 18. Pain, V. M. (1986) Biochem. J . 235,625-637 19. Benne, R., and Hershey, J. W. B. (1978) J. Biol. Chem. 253,

20. Marcus, A. (1970) J. Biol. Chem. 245, 962-966 21. Kramer, G., Konecki, D.9 Cimadevih J. M.9 and HardeStY, B. 44. Godefroy-Colburn, T., Ravelonandro, M., and Pin&, L. (1985)

(1976) Arch. Biochem. Biophys. 174, 355-358

J. Mol. Biol. 116, 755-767 46. Kozak, M. (1986) Proc. Natl. Acad. Sci. U. S. A. 8 3 , 2850-2854

3078-3087

22. Trachsel, H., Emi, B., Schrier, M. H., and Staehelin, T. (1977) 45. Pelletier, J., and Sonenberg, N. (1985) Cell 40,515-526

23. Kozak, M. (1980) J. Mol. Biol. 144, 291-304 47. Seal, S. N., Schmidt, A., Sonenberg, N., and Marcus, A. (1985) 24. Kozak, M. (1980) Cell 22,459-467 25. Duncan, R., and Hershey, J. w . B. (1985) J. Bid. Chem. 260, 48. Butler, J. S., and Clark, J . M., Jr. (1984) Biochemistry 23, 809-

26. Grifo, J. A., Tahara, S. M., Leis, J. P., Morgan, M. A., Shatkin, 49. Lax, S., Fritz, W., Browning, K., and Ravel, J . (1985) Proc. Natl. A. J., and Merrick, W. C. (1982) J. Biol. Chem. 257, 5246- 5252 50. Seal, S. N., Schmidt, A., Marcus, A., Edery, I., and Sonenberg,

27. Grifo, J . A., Abramson, R. D., Satler, C. A,, and Merrick, W. C. N. (1986) Arch. Biochem. Biophys. 246, 710-715 (1984) J. Bid. Chem. 259, 8648-8654 51. Miles Laboratories Inc. (1981) Biochemicals and Zmmunochemi-

28. Sippel, A. (1973) Eur. J. Biochem. 37, 31-40 cals, p. 56, Miles Laboratories Inc., Elkhart, IN 29. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular 52. Lawson, T. G., Ray, B. K., Dodds, J. T., Grifo, J. A., Abramson,

Cloning; A Laboratoory Manual, pp. 150-163, Cold Spring Har- R. D., Merrick, W. C., Betsch, D. F., Weith, H. L., and Thach, bor Laboratory, Cold Spring Harbor, NY R. E. (1986) J. Bid. Chem. 216, 13979-13989

Arch. Biochem. Biophys. 238, 146-153

5486-5492 815

Acad. Sci. U. S. A. 82,330-333