INTRODUCTION

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Eukaryotic translation initiation factor 2 (eIF2) is responsible for one of the earliest steps in the initiation of protein synthesis. It forms a ternary complex with the initiator Met-tRNA^Met and GTP, which then binds to the 40S ribosomal subunit associated with eIF3, forming the preinitiation 43S complex. This, in turn, binds to the 5' end of mRNAs with the aid of eIF4F and eIF4B and migrates down the message, scanning for the first AUG codon. At this point, in a reaction requiring eIF5, GTP is hydrolyzed to GDP, and eIF2 · GDP is released, along with other initiation factors. The 60S ribosomal subunit then joins the complex, and elongation of the new polypeptide chain ensues [for reviews, see references 19 and 24]. Thus, a major role of eIF2 in this pathway is to bring the charged initiator tRNA^Met to the 40S subunit. However, eIF2 is also thought to participate in the recognition of the initiator AUG codon. Evidence for this role was obtained from a genetic analysis carried out in Saccharomyces cerevisiae, where mutations in the alpha and beta subunits of eIF2 (eIF2 and eIF2) were found to allow initiation of translation to occur at a UUG codon in a HIS4 message devoid of the normal AUG initiator triplet (4, 9, 11). The His4 protein synthesized in the presence of these suppressor mutations in eIF2 contained a methionine residue at the amino terminus, indicating that the altered eIF2 was allowing a mismatched interaction between the initiator Met-tRNA^Met and the UUG codon. More recently, mutations in the gamma subunit of eIF2 (eIF2) were also found to result in initiation taking place at UUG codons (12, 22).

eIF2 is the major site of regulation of overall protein synthesis in eukaryotic cells. It is phosphorylated by specific kinases activated, for example, upon heme deprivation in reticulocytes or by double-stranded RNA in other cell types (19). In yeast, it is responsible for the regulation of amino acid biosynthesis by being the target of the Gcn2 kinase that is activated as a result of amino acid starvation (21). Phosphorylation of eIF2 on Ser⁵¹ inhibits the initiation step of translation by blocking the exchange of GDP to GTP on eIF2 catalyzed by the guanine nucleotide exchange factor eIF2B (6, 28).

The gamma subunit contains a consensus sequence for GTP binding; it has sequence homologies to the elongation factor Tu (EF-Tu) of eubacteria in a region that has been shown for this factor to bind tRNA (17). In vivo and in vitro studies have suggested that the gamma subunit might provide EF-Tu-like functions to the eIF2 complex (13, 22). Both gamma and beta subunits can be cross-linked to guanine nucleotides and to the initiator Met-tRNA^Met (1, 24).

The role played by the beta subunit in the function of eIF2 is not clear. It contains two features that might be involved in nucleic acid interactions. In the amino-terminal half of the protein there are three runs of seven lysine residues which are conserved in yeast, human, and Drosophila sequences (11, 27, 32). Except for these repeats, the sequences in this half of the protein are considerably divergent in evolutionary terms. The carboxyl half of the protein is highly conserved, especially near the C terminus, where there is a C₂-C₂ motif reminiscent of a potential zinc finger structure. However, no zinc could be detected on purified eIF2 (27), and zinc is not required for the GTP-dependent initiator Met-tRNA^Met binding activity of eIF2 (11). An extensive mutational analysis of the C₂-C₂ motif of yeast eIF2 indicated the essential role of the cysteine residues for the in vivo function of the protein, since mutations that altered these residues, changed their spacing, or removed the motif altogether abolished function (3). Mutations found in this subunit in yeast that allow the utilization of a UUG codon for protein synthesis initiation altered residues located in or adjacent to this C₂-C₂ motif (3, 11). Of 13 independently isolated suppressor alleles of the SUI3 gene, which codes for eIF2 in yeast, 6 mapped to the region encompassed by the two pairs of cysteine residues and 7 mapped to residues located immediately next to it; all mutations altered residues that are identical or conserved in the three species. eIF2 containing suppressor forms of the beta subunit were shown to have decreased levels of GTP-dependent binding of initiator Met-tRNA^Met (11). Recently, it was determined that this defect is due to an increase in the rates of intrinsic, spontaneous GTPase activity in suppressor eIF2 complexes (22).

eIF2 is also capable of binding mRNA in vitro, although the significance of this binding during the process of protein synthesis in vivo remains to be defined (14, 15). The binding to mRNA was described to be a property of the beta subunit, based on cross-linking studies of purified eIF2 from mammalian cells (14).

To better define the role of eIF2 in the process of translation initiation, and more specifically to address in detail its potential for RNA interaction, we used a purified recombinant eIF2 in RNA binding studies. We report here the ability of eIF2 to bind mRNA in vitro and show that this binding is dependent on both the amino and carboxyl domains of the protein, involving the lysine repeats and the C₂-C₂ domain. We further show that the RNA binding detected in vitro for the beta subunit is required in vivo for the function of eIF2 in yeast. Surprisingly, given the evolutionary conservation of the three lysine runs, only one of them was found to be sufficient to provide function to the protein in vivo.

	MATERIALS AND METHODS

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Strains and genetic methods. Strains BCV59 (MATa/MAT ura3-52/ura3-52 leu2-3,112/leu2-3,112 his4^UUG-306/his4^UUG-306 SUI3/sui3::URA3) and 167-3C (MAT ura3-52 leu2-3,112 his4^UUG-306 ino1-13) have been described elsewhere (3). Standard genetic techniques and media were as described elsewhere (16). YPGal and SGal media contained galactose (4%) in place of glucose.

Plasmid constructions. For the expression in Escherichia coli of eIF2 fused to glutathione S-transferase (GST), a BamHI site was created at the initiator codon of the SUI3 gene, cloned as a HindIII fragment in the HindIII site of M13-mp10, by site-directed mutagenesis (Amersham), using the oligonucleotide 5'-ACGCACGAGGGATCCTCCGAT-3'. The BamHI fragment containing the complete coding sequence of SUI3 was transferred to pGEX2T (30) digested with BamHI, creating plasmid pBE149.

Protein expression and purification. Overnight cultures of E. coli carrying the plasmids for expression of GST fusions were diluted 10-fold in LB medium with ampicillin (100 µg/ml) and incubated for 5 h at 37°C. Induction of expression of the fusion proteins was obtained by adding 1 mM isopropyl--D-thiogalactopyranoside to the cultures and incubating the cultures for 2 h at 37°C. The cells were collected, washed with phosphate-buffered saline (PBS), and resuspended in 1 ml of double-distilled water (ddH₂O). The cells were frozen for 16 h. Breaking buffer (10% sucrose, 0.2 M NaCl, 50 mM Tris-HCl [pH 7.5]) was added (3.5 ml) with 130 µl of a 10-mg/ml lysozyme solution, followed by incubation on ice for 1 h and incubation at 37°C for 6 min. The tubes were then centrifuged for 1 h at 15,300 × g. The supernatant was transferred to another tube, 5 ml of 0.85% NaCl was added, and the tube was centrifuged for 40 min at 17,000 × g. The supernatant was mixed with 1.5 ml of glutathione-agarose beads (50%) (Sigma) for 15 min on ice. The beads were washed five times with ice-cold PBS, and the fusion proteins were eluted with 50 mM Trisma base-40 mM glutathione (reduced) on ice. The proteins were concentrated to 1 ml in a Speed-Vac, and the free glutathione was removed by dialysis against 50 mM KCl-20 mM Tris (pH 7.8)-2 mM magnesium acetate (MgOAc)-6 mM 2-mercaptoethanol-1 mM phenylmethylsulfonyl fluoride (PMSF). The purified proteins were stored at 80°C. For the purification of protein 158, cell extract was prepared in the presence of the protease inhibitors aprotinin (2 µg/ml), EDTA (10 mM), PMSF (1 mM), benzamidine (1 mM), and E-64 (2.8 µM); for solubilization of the protein, Sarkosyl was added to 1.5%.

Polysome analysis. Sucrose gradients were prepared, and the cell extracts were fractionated essentially as described previously (7). Cultures were diluted 100-fold in minimal medium supplemented with the necessary amino acids and incubated at 30°C overnight to an A₆₁₀ of 0.5 to 1.0. All subsequent steps were performed at 4°C or on ice. The cells were collected by centrifugation and washed twice with lysis buffer (10 mM Tris [pH 7.5], 100 mM NaCl, 30 mM MgCl₂, 200 µg of heparin per ml, 0.2% diethyl pyrocarbonate [DEPC]). The cells were resuspended in 1 ml of lysis buffer and broken by agitation with glass beads. The supernatant was submitted to centrifugation at 3,300 × g for 5 min, and the supernatant of this step was centrifuged again for 5 min at top speed in a microcentrifuge. The final supernatant was used for the sucrose gradients. These were prepared as a step gradient consisting of five solutions (2 ml of each) of 7, 17, 27, 37, and 47% sucrose in 50 mM Tris-acetate (pH 7.0)-50 mM NH₄Cl-12 mM MgCl₂-1 mM DTT-0.1% DEPC-1 mM PMSF, applied to polyallomer tubes (14 by 98 mm; Beckman), and left at 4°C overnight. The volume of extract equivalent to 10 A₂₈₀ units was brought to 800 µl with lysis buffer and applied to the top of the gradient, which was then submitted to centrifugation in an SW41Ti rotor (Beckman) at 39,000 rpm for 3 h at 4°C. The gradient was collected with a Beckman gradient collector adapted to the top of the tubes, and the gradients were forced up by the injection of a 60% sucrose solution to the bottom of the tubes with a Bio-Rad peristaltic pump at 40% maximum speed. The gradient was monitored on a Bio-Rad Econo UV monitor at A₂₅₄; fractions of 0.5 ml were collected, precipitated on ice with 10% trichloroacetic acid, subjected to SDS-PAGE, and analyzed on Western blots (31) by the enhanced chemiluminescence (ECL) detection method (ECL kit; Amersham).

Antisera. Sera against the three subunits of eIF2 were raised in female New Zealand rabbits immunized with proteins expressed in E. coli and purified as described above. Proteins (100 to 200 µg) were injected subcutaneously with complete Freund's adjuvant; 30 and 60 days later, 100 to 200 µg of protein was injected in incomplete Freund's adjuvant. The rabbits were bled 10 days after the second and third injections.

Western blots. After SDS-PAGE, the proteins were transferred to nitrocellulose membranes (Hybond-C Extra; Amersham) (31). The membrane was blocked with 10% low-fat milk for 30 min, washed extensively with ddH₂O, and incubated with the primary antibodies in the appropriate dilutions (1:100, 1:500, and 1:200 for antibodies against the alpha, beta, and gamma subunits, respectively) in Tris-buffered saline (TBS; 150 mM NaCl, 50M mM Tris [pH 7.4]) for 60 min at room temperature. The membrane was washed in TBS-Tween 20 (0.05%) for 10 min, washed with water, and incubated for 1 h with protein A coupled to peroxidase (Sigma), diluted 1:2,000. The membrane was washed three times with TBS-Tween for 10 min and twice for 5 min in 26 mM sodium carbonate-35 mM sodium bicarbonate, followed by detection with an ECL kit (Amersham). For the immunoprecipitation studies, the bands corresponding to immunoglobulin G heavy chain bound to the beads were cut from the nitrocellulose filters after staining with Ponceau stain.

Immunoprecipitations. Agarose beads coupled to protein A (Amersham) were washed five times with coimmunoprecipitation buffer (COIP buffer; 20 mM Tris [pH 7.5], 50 mM KCl, 0.1% Triton X-100, 1 mM PMSF) and resuspended 1:1 (vol/vol) in the same buffer. Beads (5 µl) were incubated with 5 µl of serum and 190 µl of COIP buffer for 1 h at room temperature with gentle agitation. The beads were washed three times with COIP buffer; cell extracts (100 µg of total protein) were added to the beads, bringing the volume to 200 µl with COIP buffer, and incubated for 2 h at 4°C with gentle mixing. The beads were collected by centrifugation, washed three times in 200 µl of COIP buffer, resuspended in 10 µl of sample buffer, boiled for 3 min, and submitted to SDS-PAGE for immunoblotting.

RNA synthesis and labeling. A 555-bp EcoRI-BamHI fragment from plasmid pBE9, containing 53 nucleotides from the untranslated leader region and 502 nucleotides of the coding region of the HIS4 gene (8), was cloned into the XhoI and BamHI sites of plasmid pSP72 (Promega), after fill-in of the EcoRI and XhoI termini with the Klenow fragment of DNA polymerase, creating plasmid pBE155. The transcription reaction used as the template 1 µg of plasmid pBE155 linearized with HindIII and was carried out in a volume of 20 µl in the presence of 0.02 mM GTP-0.4 mM ATP-0.4 mM CTP-0.1 mM UTP-20 µCi of [³²P]UTP-0.5 mM m7GpppG (Boehringer Mannheim)-20 U of RNase inhibitor (Boehringer Mannheim)-7.5 U of SP6 polymerase-40 mM Tris (pH 7.9)-6 mM MgCl₂-2 mM spermidine-2 mM DTT for 15 min at 37°C, followed by the addition of 0.4 mM GTP and incubation for 2 h at 37°C. The template was eliminated by digestion with 2 U of DNase (free of RNase; Boehringer Mannheim) for 10 min at 37°C. The enzymes were removed by phenol extraction, and excess nucleotides were eliminated by four ethanol precipitations. The RNA was resuspended in 100 µl of DEPC-treated ddH₂O. Charged initiator tRNA^Met was prepared essentially as described elsewhere (11) except that [³⁵S]methionine (1,175 Ci/mmol; Amersham) was used; the specific activity of initiator [³⁵S]Met-tRNA^Met was 142,000 cpm/pmol.

RNA binding assays. Proteins immobilized on nitrocellulose filters (Hybond-C; Amersham) were incubated in blocking buffer (2.5 mM EDTA, 0.05% Triton X-100, 0.04% Ficoll, 0.04% polyvinylpyrrolidone) for 1 h at room temperature. The filters were then incubated in binding buffer (150 mM KCl, 20 mM Tris [pH 7.8], 2 mM MgOAc, 2.5 mM EDTA, 0.05% Triton X-100, 0.04% Ficoll, 0.04% polyvinylpyrrolidone) for 1 h at room temperature, and binding was performed in the same solution supplemented with 20 µg of denatured salmon sperm DNA per ml and RNA at 125,000 cpm/ml (average specific activity of 2.4 × 10⁶ cpm/µg) for 1 h at room temperature. After binding, the filter was washed three times in washing buffer (50 mM KCl, 20 mM Tris [pH 7.8], 2 mM MgOAc) for 10 min each and dried at room temperature, and the bound radioactivity was measured on a Molecular Imager system (Bio-Rad), using the Molecular Analyst software (Bio-Rad). The relative quantification of the proteins was done by staining the immobilized proteins with amido black, scanning the filter on an Epson ES-1000C scanner, and analyzing the intensity of the bands with the Molecular Analyst software (Bio-Rad). For solution binding assays of purified GST fusions, approximately 1 µg of each protein was incubated with 50 µl of glutathione-Sepharose beads. After being washed with STE buffer, the beads were washed with blocking buffer. Labeled mRNA (32,000 cpm) was added in 300 µl of binding buffer. After incubation for 1 h at room temperature, the beads were washed thoroughly in washing buffer, and the radioactivity retained on the beads was counted. The beads were then submitted to SDS-PAGE for quantitation of the bound proteins. The assay conditions for the determination of GTP-dependent binding of initiator Met-tRNA^Met by eIF2 or by purified GST fusion proteins were essentially as described previously (11).

	RESULTS

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RNA binding by the wild-type yeast eIF2 fused to GST. The GST-eIF2 fusion protein was isolated from E. coli as a highly purified, soluble protein and assayed for its ability to bind RNA in vitro. The RNA used in this study was derived from a 555-bp fragment of the S. cerevisiae HIS4 sequences, comprising 53 nucleotides of the leader sequence and 502 nucleotides from the coding region, transcribed in vitro from the SP6 promoter. The labeled mRNA was used for the binding assays with proteins immobilized on nitrocellulose membranes (Northwestern blots). As shown in Fig. 1, the wild-type protein fused to GST was capable of binding this RNA, while GST alone retained no radioactivity. In this figure, the fast-migrating protein stained with amido black in the GST-eIF2 preparation is a degradation product, always present in this purification procedure, composed mostly of the GST moiety; it does not interact with RNA. For these binding reactions a large excess of DNA over labeled RNA was added, and this was shown not to interfere with or compete for RNA binding; in addition, capped and uncapped RNAs were bound equally well (results not shown). This fusion protein did not bind initiator Met-tRNA^Met in solution assays (data not shown).

View larger version (46K): [in this window] [in a new window]   FIG. 1.   RNA binding by eIF2. (A) Northwestern blot of purified wild-type GST-eIF2 protein (lane 1) and GST (lane 2); (B) Northwestern blots of whole-cell extracts of bacteria induced for the expression of eIF2 (lane 1), GST-eIF2 (lane 2), and GST-eIF2 (lane 3). The proteins were resolved on an SDS-10% polyacrylamide gel, transferred to a nitrocellulose membrane, and incubated with labeled synthetic RNA as described in Materials and Methods. The immobilized proteins were visualized by staining the filter with amido black in the case of the purified proteins or by Western blotting in the case of the whole-cell extracts, using a mixture of sera directed at each individual subunit (a), and the bound radioactivity was detected by autoradiography (b).

Mapping regions in eIF2 required for RNA binding. To map sequences in eIF2 that mediate the RNA binding activity of this subunit, several truncated forms of GST-eIF2 were constructed (Fig. 2) and expressed in E. coli. The purified proteins were assayed for RNA binding on Northwestern blots. The binding activity of each protein was quantitated relative to the RNA binding displayed by the wild-type fusion and normalized for the amount of protein present on the filter relative to the wild-type fusion (Fig. 3).

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Schematic representation of the GST-eIF2 fusion deletions. Only the portion corresponding to eIF2 is shown, with amino acid residues included in the truncated proteins indicated. The wild-type protein is shown at the top, with the lysine runs depicted as hatched boxes and the C2-C2 (c.c) motif indicated. Numbering used for the proteins is identical to numbering of the expression plasmids.

View larger version (51K): [in this window] [in a new window]   FIG. 3.   Northwestern blot analysis of the GST-eIF2 deletion proteins. The purified recombinant derivatives of GST-eIF2 were submitted to SDS-PAGE (10% polyacrylamide gel) followed by transfer to nitrocellulose membranes. After incubation with labeled synthetic RNA, the radioactivity bound to the immobilized proteins was measured on a Molecular Imager, using the Molecular Analyst software. (a) Filter stained with amido black; (b) radioactivity bound to the proteins as detected by the Molecular Imager; (c) quantification of RNA binding on each protein band normalized for the amount of protein, relative to the wild type, as determined by the Molecular Analyst software from a scanned image of the amido black-stained filter. The radioactivity signal obtained for the wild-type GST-eIF2 protein was taken as 100% binding. The quantitative data was obtained from the average of at least three independent experiments, with the standard deviations shown on the bars for each protein.

Only one lysine run is required for function in vivo. The in vitro data indicating an important role for the N-terminal sequences in RNA binding, and the probable role played by the lysine repeats in this interaction, prompted us to perform a mutational analysis of this feature to address its in vivo function. Therefore, the three lysine repeats were removed, independently or in several combinations, from the wild-type SUI3 gene (Table 1). The deleted alleles were assayed for function in vivo by the ability to rescue a lethal disruption of a chromosomal copy of SUI3. They were transferred to a LEU2 centromeric plasmid and used to transform the diploid strain BCV59 to leucine prototrophy. The transformants underwent meiosis, and the viability of the spores in each tetrad was determined. The results of this analysis are shown in Table 1. Removal of any one of the lysine blocks or of any combination of two of them did not affect the function of the protein, since germination of three and four spores was obtained. All Ura⁺ spores were also Leu⁺, indicating that the source of the complementing SUI3 gene was the deleted allele present in the LEU2 plasmid. Only when the three lysine blocks were eliminated from the protein did it lose function, leading to the germination of only two spores in all tetrads dissected, all being Ura. The presence of the deleted proteins in the Ura⁺ Leu⁺ ascospores was verified by Western blotting (22a). Therefore, only one run of lysines, irrespective of its position, is sufficient to provide function to eIF2.

View larger version (29K): [in this window] [in a new window]   FIG. 4.   In vivo interactions of eIF2(K1K2K3). (A) Association with eIF2 and - determined by immunoprecipitation of eIF2 from a cell extract of strain 167-3C containing the SUI3 allele deleted of the sequences coding for the three lysine blocks present on the high-copy-number LEU2 plasmid YEp351 (pBE237) as well as the genes for eIF2 and - in the high-copy-number URA3 plasmid YEp352 (pBE188). Total yeast cell extract was incubated with antibodies raised against eIF2 (lane 1), eIF2 (lane 3), and eIF2 (lane 4) and with preimmune serum (lane 2), adsorbed to agarose-protein A beads. The material bound to the beads was submitted to SDS-PAGE (10% polyacrylamide gel); the proteins were transferred to a nitrocellulose membrane and visualized by Western blotting with antibodies directed to eIF2. (B) Association with ribosomal subunits. Fractions (1 through 13) obtained from a sucrose gradient of a cell extract from strain 167-3C/pBE237, prepared in the absence of cycloheximide, were subjected to Western blot analysis using antibodies directed to eIF2. The peaks corresponding to the ribosomal subunits (40S and 60S) as well as the monosomes (80S) are indicated; E, cell extract before the gradient.

View larger version (37K): [in this window] [in a new window]   FIG. 5.   Suppression of initiator codon mutation by eIF2 lacking two lysine repeats. Transformants of strain 167-3C, containing centromeric plasmids carrying the SUI3 gene with the indicated mutations, were grown in replica on plates with minimal medium containing histidine (+HIS) or lacking histidine (HIS). WT, wild type.

In vitro RNA binding by eIF2 requires at least one run of basic residues. The results of the in vivo experiments prompted us to analyze the deleted proteins for the ability to bind RNA. Since the lack of all lysine repeats led to a nonfunctional protein, a GST fusion of this protein (protein 242) was purified and first assayed on a Northwestern blot for its RNA binding activity (Fig. 2). This protein was completely unable to bind RNA (Fig. 3). No radioactivity was detected even after prolonged exposure of the filter. The complete inability of this protein to bind RNA was unexpected, given that proteins lacking all N-terminal sequences (proteins 191, 192, and 193) still show low levels of RNA binding, associated with the C-terminal half. The increase in binding observed when the central portion of the protein was removed indicated that these sequences in some of the constructs may have been hindering the refolding of the protein on the nitrocellulose filter, thus perhaps causing the lack of RNA interaction observed for protein 242. Therefore, mRNA binding assays were performed in solution with the fusion proteins coupled to glutathione beads (Fig. 6). In this assay, the protein lacking the three lysine residues was able to bind RNA at 26% of the wild-type level, a result consistent with the RNA binding activity provided by the C-terminal sequences as measured on the Northwestern blots and confirmed for protein 193 in solution binding assays (data not shown). The protein containing only K3 and those derivatives in which K3 was replaced by R3 or A3 were also expressed in and purified from E. coli as GST fusions and then tested for RNA binding in solution (Fig. 6). The presence of only one lysine block increased binding to approximately 80% of the wild-type value, and the arginine run increased binding to wild-type values, whereas the alanine substitution kept the low RNA binding activity of the protein lacking all three lysine repeats. The stronger binding provided by the arginine run relative to that provided by the lysine run may reflect its higher potential for interacting with nucleic acids and further attests to the importance of the lysine motifs, and not another feature in the N-terminal region, in maintaining the interaction with mRNA. These results are strong evidence for the role played by the lysine repeats in RNA recognition and binding by eIF2. Furthermore, they reflect the in vivo results for cell viability.

View larger version (44K): [in this window] [in a new window]   FIG. 6.   Requirement of one run of charged residues for mRNA binding. Approximately 1 µg of each fusion protein, coupled to glutathione-Sepharose beads, was assayed for binding of labeled mRNA. The input of mRNA was 32,000 cpm, and the wild-type (WT) protein typically bound 10% of the label. Binding activities are given as percentages of the wild-type level and are averages of three independent experiments. The relative values were calculated by quantitating the amount of proteins present on the beads on an SDS-polyacrylamide gel. The stained gel was scanned and analyzed with the Molecular Analyst software.

Binding of mRNA, but not Met-tRNA^Met, by eIF2 requires the lysine runs. We next examined whether the ability of the eIF2 complex to bind mRNA was dependent on the lysine blocks of the beta subunit. The GST-eIF2 protein was expressed in yeast from a centromeric plasmid under the control of the galactose-inducible GAL1-CYC1 promoter (plasmid pBE195). The function of the fusion protein in vivo was determined by its ability to allow growth of cells containing a disrupted SUI3 gene. Plasmid pBE195 was introduced into the diploid strain BCV59, and the transformants underwent meiosis. The asci were dissected on plates containing either glucose or galactose. In the presence of glucose, the diploids gave rise to only two viable spores, as expected, since SUI3 is an essential gene and the expression of the GST-eIF2 fusion is repressed. When dissected on plates containing galactose, three or four viable meiotic products were obtained from the asci (data not shown).

View larger version (24K): [in this window] [in a new window]   FIG. 7.   mRNA and initiator Met-tRNAMet binding by mutant eIF2. The eIF2 complex purified from yeast cells on glutathione beads was coupled to anti-eIF2 antibodies on protein A-Sepharose beads, and these beads (30 µl) used in the mRNA binding assays. The input of mRNA was 181,000 cpm, and the wild-type (WT) complex retained typically 39,500 cpm. Binding activities, given as percentages of the wild-type level (100%), were normalized according to the amount of eIF2 complexes on the beads. For this quantitation, aliquots of the eIF2-protein A-Sepharose beads used in each assay were subjected to Western blotting using antibodies directed against eIF2, a subunit that is not involved in any step of the purification procedure and thus should be present only as a result of its association with the other two subunits. An example of this Western blot is shown next to the graph, which includes the preparations obtained from cells expressing GST-eIF2 (WT), GST-eIF2(K1K2K3) (del), and nonfused GST (GST). Since the serum raised against the gamma subunit also contained anti-GST antibodies, it was extensively adsorbed with purified GST to eliminate reaction against the GST-eIF2 fusion protein present in this assay. No free GST-eIF2 was present on these beads, as judged from the relative intensities of signals obtained from reprobing this blot with anti-eIF2 serum (data not shown). For the GTP-dependent Met-tRNAMet binding, the eluate (10 µl) from the glutathione-Sepharose beads was used directly in filter retention assays. The input of labeled Met-tRNAMet was 160,000 cpm. The counts retained on filters in the reaction lacking GTP were subtracted from the counts retained in the presence of GTP (1.2 mM). Quantitation of the complex present on the eluate was obtained by Western blotting using anti-eIF2 antibodies. GTP-dependent binding activity increased linearly with the increase in either eluate volume or labeled tRNA, and only the values of a representative point are given. The control used in both mRNA and Met-tRNAMet binding assays was an identical preparation obtained from a yeast strain containing the vector pEG(KG) expressing GST. The results shown are averages of at least three independent experiments, with variation of less than 10%.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

This work provides direct evidence for the mRNA binding activity of eIF2. The deletion analysis performed here indicates a crucial role for the lysine residues and the C₂-C₂ motif in providing strong binding of eIF2 to RNA. The results showing that the removal of the lysine repeats resulted in the inability of the protein to rescue a lethal mutation in SUI3 indicate that the lysine residues are also required in vivo. Furthermore, the fact that a run of arginine, but not alanine, residues can recover wild-type levels of mRNA binding in vitro and function in vivo provides strong evidence for the physiological relevance of this interaction. Motifs rich in basic residues, mainly arginine, present in a number of eukaryotic and prokaryotic proteins, such as the human immunodeficiency virus Tat and Rev proteins, antiterminators of phages lambda and P22, and ribosomal proteins, have been shown to direct binding of neighboring residues on these proteins to specific sites on their RNA targets (2, 5, 18, 23). Arginine-rich motifs in the translation initiation factor eIF4B have also been implicated in RNA binding (25). eIF2, however, is unusual in that it is the only case where a polylysine run has been found to be involved in RNA binding.

Because no other conserved sequences are found in the N-terminal portion of eIF2, it is reasonable to assume that the lysine clusters in eIF2 are probably required for a nonspecific interaction with the backbone of an RNA component of the translation initiation apparatus. This motif might therefore function as a facilitator for a secondary binding through the sequences located in the carboxyl half of eIF2.

The finding that the triple-deletion mutant still associates with the 40S ribosomal subunit even though it is unable to bind RNA efficiently in vitro argues against a role of the lysine runs in providing an interaction of eIF2 with rRNA. This result also makes it unlikely that the lysine blocks are involved with initiator Met-tRNA^Met binding, because 43S complexes can form only in the presence of the complete ternary complex (24). Indeed, we have shown that eIF2 complexes lacking the lysine residues bind this tRNA species as well as the wild type. Therefore, these charged clusters might serve in a later step in the process of translation initiation, such as in the binding of mRNA, as the specificity of these in vitro binding assays indicates.

The involvement of the lysine repeats with a strong mRNA binding activity is in contrast to cross-linking data for intact eIF2, where only the C-terminal portion of the beta subunit was found to be covalently bound to mRNA (14). It is possible that the interaction of mRNA with the lysine blocks of eIF2 in the intact eIF2 factor in vivo occurs during a specific point during the translation initiation process which was not mirrored in the cross-linking experiments, such as after the joining of some other factor(s) which would have to be present in our eIF2 preparation. This is a rather intriguing possibility, given that eIF2 is known to be involved in many allosteric interactions. However, it cannot be ruled out that the physical proximity of the N-terminal region of the beta subunit and mRNA could not be detected by the UV-induced cross-linking method used by Flynn et al.

The mRNA binding region detected in this work involving the C-terminal domain of eIF2 agrees entirely with the cross-linking data obtained with purified eIF2 (14). From the results shown in this work, a large segment of the carboxyl half of eIF2, including the C₂-C₂ motif, seems to participate in this interaction. The C₂-C₂ region of eIF2 is apparently involved in interactions with GTP and the initiator Met-tRNA^Met, as suppressor mutations that map in this region lead to eIF2 complexes with a high intrinsic rate of spontaneous GTP hydrolysis and to a high rate of initiator Met-tRNA^Met dissociation (22). Because the gamma subunit contains sequence motifs suggestive of a tRNA binding element, as deduced from its similarity to EF-Tu, the interaction with Met-tRNA^Met might be an intrinsic property of this subunit. However, the beta subunit, through its C-terminal portion, could also compose the Met-tRNA^Met binding pocket. Evidence from Donahue's group indicates that in the presence of a nonhydrolyzable GTP analogue, eIF2 containing a suppressor beta subunit binds the initiator Met-tRNA^Met with lower affinity than the wild-type eIF2 complex, suggesting that eIF2 participates in the binding to this tRNA species (22). Even though we did not detect binding of the initiator Met-tRNA^Met to the purified beta subunit, the RNA binding activity of the C-terminal half of eIF2 may reflect a potential Met-tRNA^Met pocket of the eIF2 complex, with the specificity being given by the interaction with the other subunits. Alternatively, as the available data taken together indicate, the C₂-C₂ region of eIF2 would make the codon-anticodon interaction possible by providing a scaffold for the binding of mRNA and the initiator tRNA, with the latter being mostly, but not completely, maintained in the appropriate position by a specific interaction with the gamma subunit. The participation of this region in interacting with eIF2 is suggested by the observation that the deletion of the C₂-C₂ motif impaired the association of the mutant eIF2 with the other two subunits of eIF2, as determined from coimmunoprecipitation studies (26a). For this reason, we could not address in more detail the participation of this motif in mRNA binding by the eIF2 complex. However, we have performed mRNA binding assays with the C-terminal half of eIF2 proteins containing different suppressor mutations and found no difference relative to the wild-type protein (data not shown). This observation indicates that the positions altered in these mutant proteins are not primarily involved in establishing interaction with mRNA and supports the observations by Donahue's group that these mutant eIF2 complexes have impaired tRNA binding activities (22).

It is interesting that only one block of lysine residues was found to be required for function, both in vitro and in vivo. We have shown that the single deletions and the double deletions, except the one which leaves only K2, do not derepress translation of GCN4, an assay that is widely used to detect defects in translation initiation factors, including eIF2 (22a). This lack of any deleterious effect when most of the lysine runs are missing is surprising, considering the evolutionary conservation of the three copies of this motif. It is possible that they are required under growth conditions that have not been tested and that do not cause derepression of GCN4 translation.

It has recently been reported that K2 of eIF2 is required for the interaction of eIF2 with eIF5 (10). Our results showing that all single or double deletions involving the lysine runs result in functional proteins, taken together with data indicating that eIF5 is an essential protein in yeast, suggest that the interaction between the two factors must occur elsewhere. The interaction of eIF5 with eIF2 may occur through sequences neighboring K2, which are made inaccessible for interaction by the deletion of the lysine residues in the in vitro conditions used by that group. It is clear, then, that a more detailed study of the sites of interaction between eIF5 and eIF2 in vivo is needed. The indications that eIF5 interacts with eIF2 through the amino-terminal region may suggest an alternative mechanism by which the suppressor form of eIF2 containing only one lysine run is not able to initiate translation at a UUG codon as efficiently as the suppressor protein containing all three lysine repeats. It has been recently demonstrated that this same suppressor mutation confers to the eIF2 complex an increased intrinsic GTPase activity, which might lead to a premature hydrolysis of GTP when the scanning ribosome encounters a UUG codon, therefore initiating translation at this triplet (22). If interaction with eIF5 is defective in eIF2(K1K2), eIF5-dependent GTP hydrolysis will also be deficient, thus decreasing the efficiency of utilization of UUG codons by the suppressor derivative. It is reasonable to suppose that both a defective interaction with eIF5 and a decreased affinity for mRNA could cause the near inability of this deleted protein to allow initiation of translation at a UUG codon. The hypothesis of a defective, but not null, eIF5 interaction with proteins lacking K1 and K2 would also explain the total lack of suppression activity by eIF2(K1K2)R3, which is capable of binding mRNA in vitro at wild-type levels.

This work provides strong evidence for mRNA binding by eIF2. Previous data regarding a potential ability of eIF2 to interact with RNA were based on cross-linking studies, which can determine only physical proximity. Biochemical analyses of the RNA binding by eIF2 always relied on preparations of this complex, which can be contaminated with other factors and often contain degradation products of the beta subunit, which is notoriously unstable. Therefore, the binding data shown here are relevant to the characterization of a possible role of eIF2 in the formation of the initiation complex in eukaryotes.