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Previous Article | Next Article 
Molecular and Cellular Biology, January 2003, p. 687-698, Vol. 23, No. 2
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.2.687-698.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Microbiology and Immunology, State University of New York Downstate Medical Center, Brooklyn, New York 11203,1 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia2
Received 10 July 2002/ Returned for modification 9 September 2002/ Accepted 11 October 2002
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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450 nucleotides (nt) long and comprises H, I, J-K, and L structural domains upstream of the initiation codon AUG at nucleotide 834 (AUG834). Ribosomal initiation complexes attach directly to AUG834, and initiation does not involve scanning (19, 35). Recent analysis has begun to resolve molecular details of the mechanism of initiation on EMCV-like IRESs. Viral and cellular IRES-containing mRNAs can be translated under conditions when the cap-mediated mode of end-dependent initiation that is used by most mRNAs is inhibited (12), and studies of initiation on viral IRESs are therefore yielding insights into mechanisms that enable selective translation of mRNAs in the cell.
The stage that differentiates IRES-mediated initiation from the conventional cap-mediated mode of initiation is the mechanism by which the 43S ribosomal preinitiation complex engages mRNA to form a 48S complex at the initiation codon (16, 36). The 43S complex consists of a 40S ribosomal subunit, initiator tRNA, and eukaryotic initiation factors (eIFs), including eIF2 and eIF3. In the process of cap-mediated initiation, eIF4F is thought to create a binding site for this complex adjacent to the 5'-terminal m7G cap by inducing local conformational changes in bound mRNA (42) and by enhancing the binding of the eIF3 component of 43S complexes to mRNA (5). The 43S complex binds to the mRNA through a network of protein-protein and protein-RNA interactions and then scans to the initiation codon. Many of these interactions involve eIF4F, which comprises the cap-binding protein eIF4E, the RNA helicase eIF4A, and either the eIF4GI or eIF4GII isoform. The amino-terminal one-third of eIF4G (amino acid residues 1 to 675) binds eIF4E and the poly(A) binding protein (10, 24). The central one-third of eIF4G, which binds to eIF3 and contains one of the two eIF4A-binding sites, consists of five pairs of 
-helices that are known as HEAT repeats (29).
Biochemical reconstitution of 48S complex formation from purified components on the EMCV IRES has shown that this process is ATP dependent and that it requires the same initiation factors as cap-dependent initiation, except for eIF1, eIF1A, and intact eIF4F (34, 35, 38). Similar results have been described for FMDV and TMEV IRESs (40). 48S complexes assembled in this way are competent to complete all remaining steps in initiation (T. V. Pestova, unpublished data). Initiation on the EMCV IRES has no requirement for eIF4E, and eIF4F can be replaced by eIF4A and a central domain of eIF4G, namely, eIF4GI, located from amino acid residues 737 to 989 (eIF4GI737-989) (26). The requirement for eIF4A and eIF4G is consistent with the profound inhibition of EMCV and FMDV translation by dominant-negative mutant forms of eIF4A, such as the R362Q substitution mutant, which sequester the eIF4A/eIF4G complex in an inactive form (33, 47). Initiation on the EMCV IRES does not involve scanning, and the basis for the requirement for ATP and for active eIF4A for initiation on the IRES is not known. eIF4A binds to the central -helical domains of eIF4GI737-989 and eIF4GII745-1003 and greatly enhances their affinity for the IRES in an ATP-independent manner (26, 29).
In addition to requiring canonical initiation factors, assembly of 48S complexes on some picornavirus IRESs is enhanced by IRES trans-acting factors that bind to defined sites on them and induce conformational changes in these IRESs that facilitate binding of the eIF4G/eIF4A complex (3, 20, 35, 37, 40). IRESs are therefore not static scaffolds but undergo conformational transitions as they bind components of the translation apparatus during the initiation process. Initiation on the IRES of the EMCV strain used in the experiments described here does not require IRES trans-acting factors (35). However, the EMCV IRES becomes dependent on the pyrimidine tract-binding protein as a result of mutations either in the domain of the IRES to which eIF4G binds or in the coding region adjacent to the initiation codon (20).
EMCV-like IRESs have conserved structures that are thought to orient binding sites for components of the translation apparatus to promote recruitment of the 43S complex and consequent assembly of the 48S complex (17, 37). eIF4G binds specifically to the J-K domain of these IRESs, and this interaction is necessary for initiation (22, 26, 27, 29, 38, 40, 46). The binding site on the EMCV IRES has been localized by footprinting (22); residues are protected in the A-rich loop at the junction of the J and K domains and in the flanking J2, J3, and K1 helices (Fig. 1). Deletion of nucleotides in the A-rich loop severely reduces both binding of eIF4G/eIF4A and IRES-mediated initiation (35). Although the J-K domain is essential, it is by itself not sufficient for EMCV IRES function, and experiments to date account only for the function of the central core of this domain. The extensive sequence conservation in EMCV-like IRESs extends over much of the J-K domain (18), including regions such as the terminal loops of the J and K domains and the J3 helix that have been found to be important for initiation (13, 32, 48). The adjacent domain I also plays an essential but as yet undefined role in EMCV IRES function, and the flanking domain H, domain L, and sequences downstream of domain L all also play accessory roles (7, 9, 18, 20). The molecular basis for the importance of these regions of the IRES has not yet been determined.
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MATERIALS AND METHODS |
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Plasmids. EMCV pTE1 (9), pTE3-GUS (2), and pJK (26) transcription vectors and vectors for expression of recombinant wild-type eIF4A (35), R362Q mutant eIF4A (37), eIF4GI737-1116, eIF4GI737-1009, and eIF4GI737-1600 (26) and eIF4GII745-1003 (29) have been described previously. The eIF4GI plasmids have been renamed to take into account a recent revision to the sequence of the largest eIF4GI isoform, extending its amino terminus by 40 amino acid residues (4). Deletion mutants of the EMCV IRES were generated in pTE3-GUS and pJK plasmids by using a two-step PCR. Substitution mutations in eIF4GI737-1116 were made exactly as described previously (26). All mutations were confirmed by sequencing the complete IRES or the eIF4GI737-1116 coding sequence, as appropriate.
In vitro translation. mRNA comprising the EMCV IRES linked to the ß-glucuronidase open reading frame was transcribed by using T7 RNA polymerase and was translated in RRL in accordance with the manufacturer's instructions in the presence of [35S]methionine. Translation products were resolved by electrophoresis with 12% polyacrylamide gel. Gels were dried and exposed to X-ray film. The efficiency of translation was quantified by using a Molecular Dynamics PhosphorImager.
Assembly and analysis of ribosomal complexes. Ribonucleoprotein and ribosomal 48S complexes were assembled on EMCV RNAs and analyzed by primer extension by using the primers 5'-GTCAATAACTCCTCTGG-3' (complementary to EMCV nt 957 to 974) and 5'-GGGGGATGTGCTGCAACC-3' (complementary to nt 368 to 351 in the polylinker of pTZ18R), as appropriate, and AMV-RT in the presence of [32P]dATP, as described previously (26, 35, 38).
Footprinting analysis of initiation factor-IRES complexes. Free or initiation factor-bound RNAs in binding buffer were probed with DMS or RNase V1, and chemically modified or enzymatically cleaved RNAs were analyzed by reverse transcription, as described previously (21, 22, 40). ATP was present in all probing reactions that contained eIF4A or eIF4F. Hydroxyl radical footprinting was done essentially as described previously (15).
Preparation of Fe(II)-BABE-derivatized eIF4G. Cysteine-containing mutants of eIF4GI737-1116 (0.2 to 0.5 µg/µl in 95 µl of buffer containing 80 mM K+-HEPES [pH 7.6], 300 mM KCl, and 10% glycerol) were conjugated with 5 µl of 20 mM Fe(II)-bromoacetyl-amidobenzyl-EDTA (BABE) (Dojindo Molecular Technologies, Inc., Gaithersburg, Md.) by incubation at 37°C for 30 min essentially as described previously (6). Unincorporated Fe-BABE was separated from derivatized eIF4G by loading reaction mixtures onto pretreated Microcon YM-30 microconcentrators and washing four times with the incubation buffer. Fe-eIF4G samples were typically recovered in 50 to 80 µl of buffer. The accessibility of each introduced cysteine residue for derivatization and the efficiency of the Fe(II)-BABE tethering reaction were assessed by using the reaction with the thiol-specific fluorescent coumarin derivative DCIA (Molecular Probes, Eugene, Oreg.) as described previously (6).
Directed hydroxyl radical probing. Ribonucleoprotein complexes were assembled by incubating 0.2 µg of EMCV RNA (nt 280 to 974) in 40 µl of buffer containing 80 mM K+-HEPES (pH 7.6), 300 mM KCl, 2 mM MgCl2, and 10% glycerol with 0.8 to 1 µg of Fe-eIF4G (and, where indicated, 1 µg of eIF4A) at 37°C for 5 min and then placing them on ice. Fenton chemistry was initiated by the addition of 2 µl of a freshly prepared mixture of 0.1 M ascorbic acid and 0.5% H2O2 to each sample to generate hydroxyl radicals in the vicinity of the tethered Fe(II). Reactions were quenched after incubation for 10 min in ice by the addition of 20 µl of 20 mM thiourea. EMCV RNA was isolated immediately by phenol extraction followed by ethanol precipitation, and sites of hydroxyl radical cleavage were located by primer extension analysis with the primer 5'-CGGTATTGTAGAGCAGAGC-3' (complementary to EMCV nt 854 to 836) and AMV-RT in the presence of [32P]dATP.
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RESULTS |
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J5, IRES-K2', IRES-K2 and IRES-J5K2' (Fig. 1). Chemical probing and toe printing of these RNAs in the absence of initiation factors (see below) indicated that these mutations did not alter the IRES secondary structure except in the immediate vicinity of the deletions. IRES-mediated translation of a ß-glucuronidase (GUS) reporter was almost completely abolished by deletion K2 (Fig. 2, lanes 9 and 10), whereas the smaller K2' deletion reduced translation by half (lanes 3 and 4). Deletion J5 reduced IRES-mediated translation to 15% of the level of the wild-type IRES (lanes 5 and 6), and translation was synergistically impaired by combining deletions J5 and K2' (compare lanes 7 and 8 with lanes 3 and 4 and lanes 5 and 6). These results confirm the importance of J5 and K2 hairpins for IRES function (13, 32, 48).
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K2' deletion, more by the J5 deletion, and almost completely by the K2 deletion (Fig. 3A, lanes 2, 4, and 6, and Fig. 3B, lane 1). Binding of eIF4GII745-1003 to the J-K domain was similarly reduced by these deletions (e.g., Fig. 3D and E, lanes 4), consistent with previous reports that determinants for binding of eIF4GI and eIF4GII isoforms to the IRES are similar (26, 29). Combining J5 and K2' deletions had a synergistic effect, abolishing the binding of eIF4F so that no RT stops were detected on this mutant RNA other than those apparent in the absence of eIF4F (Fig. 3A, lane 8). The effects of these mutations on the binding of eIF4F/eIF4G therefore exactly paralleled their effects on IRES-mediated translation.
Footprint analysis of mutant EMCV IRES J-K domain/eIF4G complexes.
Binary complexes of eIF4GII745-1003 and wild-type, J5, or K2' mutant EMCV RNAs (nt 680 to 787) were probed with DMS, which reacts with unpaired adenine residues and to a lesser extent with unpaired cytosine residues (8). In these experiments, several residues that appear to have altered susceptibility to cleavage or modification in the presence of factors coincide with strong stops formed during primer extension by RT on this highly structured RNA in the absence of factors. Protection or enhanced modification of these nucleotides is therefore considered to be equivocal and is not discussed. In addition to the previously described protection of AA687-688, A724, and AAAAA770-774 (Fig. 1) (22), eIF4GII745-1003 and eIF4GI737-1600 also protected A704, AC801-802, and C869 (Fig. 4A, lane 2 and Fig. 4B, lane 5). Protection at these sites was weakly but reproducibly observed. The site on the IRES to which eIF4G binds is therefore more extensive than previously realized and includes residues whose conformation is directly altered in mutant J5. eIF4GII745-1003 protected AAAAA770-774 and AC801-802 in J5 and K2 mutants from DMS modification as seen for wild-type RNA, but protection at AA687-688 was reduced, particularly for the J5 mutant (Fig. 4A, lanes 2, 5, and 8). Protection at A704 was abolished for the J5 mutant.
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Hydroxyl radical probing of EMCV IRES/eIF4G complexes. To further characterize the eIF4G binding site on the IRES, binary RNA/protein complexes were probed with hydroxyl radicals generated from free Fe(II)-EDTA. Hydroxyl radicals attack the ribose moiety in single- and double-stranded RNA in a sequence-independent manner, leading to scission of the RNA backbone (25), and this broad specificity permitted probing of the complete IRES. eIF4GI737-1600 protected extensive regions in the J, K, and L domains from cleavage (Fig. 5, lanes 2 and 3) in a manner that corresponded directly to sites of protection from RNase V1 cleavage. The protection of these helical regions of the IRES by eIF4G from cleavage by hydroxyl radicals suggests that eIF4G interacts with the minor groove of the RNA duplexes. The only site of significantly enhanced cleavage due to binding of eIF4GI737-1600 was at AA678-679 at the junction of the I and J-K domains. These results are summarized in Fig. 1.
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Mapping the orientation of eIF4G on the IRES by directed hydroxyl radical probing.
The core domain of eIF4G that is necessary and sufficient to support initiation on the IRES consists of five stacked pairs of -helical repeats, numbered 1a-1b, 2a-2b, etc. (29) (Fig. 6D). We used directed hydroxyl radical probing to orient this core domain of eIF4GI on the IRES. In this approach, based on methods used to map the binding sites for factors and ribosomal proteins on prokaryotic rRNA (6), Fe(II) tethered to a specific site on the surface of eIF4G is used to generate hydroxyl radicals to cleave the IRES.
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Fe(II) was then tethered to the protein via BABE. The activity of each Fe(II)-conjugated mutant polypeptide was comparable to the activity of unmodified wild-type eIF4GI737-1116 in binding to the IRES and in supporting 48S complex formation on the IRES in vitro (Fig. 6A and data not shown). To identify the nucleotides in the IRES that are in close proximity to bound eIF4GI, Fe(II)-conjugated polypeptides were bound to the IRES in the presence and absence of eIF4A (which enhances eIF4G's interaction with the IRES [26]). Hydroxyl radicals were then generated to cleave the IRES in the vicinity of the tethered Fe(II). Sites of cleavage mapped by primer extension (Fig. 6B) are indicated on the secondary structure of the J-K domain (Fig. 6C). Three of the eight mutants reproducibly caused specific and characteristic cleavages in the IRES that were all restricted to the J-K domain. The T830C mutation is located in the long, crystallographically disordered loop between helices 2b and 3a. The eIF4GI737-1116 T830C mutant induced weak cleavage at AA699-700 and AUC724-6 on opposing sides of the J3/J4 helical junction, and at UAU713-715; cleavage at AA699-700 and particularly at AUC724-726 was strongly enhanced by eIF4A (Fig. 6B, lanes 2 and 7). The D929DC insertion is located immediately after helix 4a, and it induced cleavage at UAU713-715 that was strongly enhanced by eIF4A (Fig. 6B, lanes 3 and 8). The eIF4GI737-1116 (C920A, C935A, C937A) substitution mutant retains C820 and C822 residues in helix 2b and C848 in helix 3a. The cleavage that this mutant induced at GCGU775-778 was enhanced by eIF4A (Fig. 6B, lanes 5 and 10). Mutant 3 retains only a single cysteine residue (C848) and did not induce cleavage at this site (data not shown). These results suggest a model for the eIF4GI737-1116/IRES complex (Fig. 6C and D) in which the N terminus of this domain of eIF4G is orientated towards helix J1 of the J-K domain and the C terminus of this domain is oriented towards helix J5.
Footprinting complexes of eIF4G, eIF4A, and eIF4B on the EMCV IRES. eIF4A, eIF4B, and eIF4G interact synergistically to form a ribonucleoprotein complex on the IRES of EMCV and related viruses (26, 38, 40). We used RNA footprinting to compare the interactions of this complex and of eIF4G alone with the EMCV IRES. eIF4A enhanced eIF4GI737-1600's protection of the IRES from RNase V1 cleavage at GUCU777-780, reduced protection at CUU812-814, and enhanced cleavage at CA674-675 at the base of domain I, at G766 and CC784-785 in the J-K domain, and weakly at UG897-898 downstream of the initiation codon AUG834 (Fig. 4B, lanes 7 to 9). Some of these changes, for example at CA674-675 and G766, were slightly enhanced by eIF4B. These changes were also observed when the R362Q trans-dominant mutant form of eIF4A replaced the wild-type factor (data not shown). eIF4A and eIF4B did not significantly alter the pattern of DMS modification of RNA in the eIF4GI737-1600/IRES complex (Fig. 4B, lanes 3 to 5). These results are summarized in Fig. 1.
ATP-dependent conformational changes in EMCV RNA downstream of the initiation codon induced by eIF4F. The ATP dependence of initiation on the IRES and the requirement for active eIF4A (33, 35) suggest that ribosomal loading onto the initiation codon AUG834 may involve restructuring of the IRES by the eIF4G/eIF4A complex. Our probing analyses (22) (Fig. 4B) indicated that sequences immediately downstream of the J-K domain are not extensively unwound by eIFs 4A, 4B, and 4G but that some conformational changes are induced in structures flanking and downstream of the initiation codon.
ATP-dependent conformational changes downstream of the initiation codon AUG834 induced by eIF4G/eIF4A were also identified by toe printing done by using EMCV RNA (nt 378 to 1155, comprising the entire IRES and 322 nt of the adjacent coding region), eIF4A, and eIF4GI737-1116 (which contains only one of the two eIF4A-binding sites in eIF4G [Fig. 7]). Binding of eIF4GI737-1116 in the presence of eIF4A enhanced stops at CGC865-867 in a manner that was further enhanced by but did not depend on the presence of ATP or on ATP hydrolysis (lanes 3 and 6). Additional new toe prints appeared at C841, A858 and (more weakly) G862, and G898 only in the presence of eIF4F, wild-type eIF4A, and ATP (lanes 7 and 12) or eIF4GI737-1116, wild-type eIF4A, and ATP (lane 5). All but one of these stops occur in a proposed irregular stem-loop structure (14) encompassing nt 854 to 902 (Fig. 1). These stops were not detected in reactions lacking eIF4A (lanes 2 and 3). They were only weakly apparent in the absence of ATP (lanes 4 and 11) or on inclusion of AMP-PNP (a nonhydrolyzable ATP analog) in place of ATP (lane 6). However, this result must be treated cautiously because these reactions also contained dATP (an essential constituent of primer extension reactions), which can partially substitute for ATP in eIF4A-dependent initiation on the EMCV IRES [38]). Significantly, these additional stops were not detected in reactions in which R362Q trans-dominant mutant eIF4A replaced wild-type eIF4A in the presence or absence of ATP (lanes 8 and 9). This eIF4A mutant is unable to utilize ATP or dATP to mediate conformational changes in RNA. Inclusion of R362Q mutant eIF4A in toe printing reactions with eIF4GI737-1116 enhanced the toe prints at C786 and CGC865-867 to a similar extent as when wild-type eIF4A was present (compare lanes 2, 4, and 8 and lanes 5 and 9). These results indicate that mutant eIF4A was incorporated into the eIF4G/eIF4A complex on the J-K domain but that the resulting complex was unable to induce ATP-dependent conformational changes in the IRES/factor complex downstream of the initiation codon.
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DISCUSSION |
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eIF4G recognizes a large and structurally complex binding site on the EMCV IRES that encompasses sequences in the J, K, and L domains. This conclusion is based on the results of hydroxyl radical and chemical and enzymatic probing (Fig. 4A, 4B, and 5A). We have previously reported that interaction of eIF4G, either alone or as part of eIF4F, with the IRES and consequent initiation of translation require the oligo(A) loop at the junction of the J and K domains (35). We have now found that binding eIF4G and consequent initiation also depend on the apical J5 and K2 hairpins, which act synergistically in these processes (Fig. 2 and 3). Taken together with observations that eIF4G/eIF4F binds specifically to the J-K domains of human parechovirus type 1 (our unpublished data), FMDV, and TMEV IRESs (27, 28, 40, 46), these findings suggest that the sequence conservation in the central region of the J-K domain and the extensive but covariant sequence differences in these peripheral helices of the J-K domain (17) are indicative of a need to maintain its structural integrity to enable eIF4G to bind. This requirement would account for the deleterious effects of mutations in these apical helices on EMCV IRES function noted here and elsewhere (13, 14, 32, 48).
The central domain of eIF4G that binds specifically to the J-K domain consists of five -helical HEAT repeats: eIF4G is the first HEAT-repeat polypeptide that is known to bind specifically to nucleic acid (29), and the results reported here therefore define aspects of a new mode of RNA-protein interaction. The HEAT-repeat domain of eIF4G binds directly to a small number of bases in the J-K domain, most significantly in the A-rich bulge at the junction of the J2, J3, and K1 helices (Fig. 1), but hydroxyl radical probing indicated that eIF4G bound more extensively to RNA duplexes in the J, K, and L domains (Fig. 1 and 5A). Hydroxyl radicals cleave the sugar-phosphate backbone of RNA at ribose C1' and C4' positions (25), so the observed protection indicates that eIF4G binds in the minor groove of these RNA duplexes. Other polypeptides that bind double-stranded RNA, such as the double-stranded RNA (dsRNA)-binding domain of proteins such as Staufen, Xlrbpa, and the dsRNA-dependent protein kinase PKR (1, 41, 45), all do so through its wide minor groove. The functional 2'-hydroxyl and phosphate groups in the RNA minor groove do not allow sequence-specific recognition, and these proteins show no sequence specificity in their interactions with RNA in vitro. A similar ability of eIF4G to bind dsRNA in a sequence-independent manner could bear on its activity as a cofactor that enhances the ATP-dependent RNA helicase activity of eIF4A (43, 44), for example by reducing dissociation of eIF4A from duplex RNA during cycles of step-wise unwinding of RNA.
eIF4B also binds directly to EMCV-like IRESs (28, 31, 38), and footprinting data (Fig. 4B) indicate that it weakly enhances binding of the eIF4G/eIF4A complex to the EMCV IRES. This conclusion is consistent with previous results obtained by using EMCV and FMDV IRESs in UV cross-linking and toe printing assays (38, 40). However, this enhancement is weak, so it may be a less important contributing factor in eIF4B's activity in enhancing ribosomal binding to this group of IRESs (35) than the interaction of eIF4B with the eIF3 component of the 43S complex (30).
eIF4GI and eIF4GII both bind specifically to the J-K domain and recruit eIF4A, which by itself does not bind specifically or stably to the IRES. eIF4A interacts strongly with the IRES in the resulting heterotrimeric complex and also significantly enhances eIF4G's binding to the IRES (22, 26, 38). The eIF4G/eIF4A complex induced limited local conformational changes in the immediate vicinity of its binding site on the IRES (Fig. 4B), suggesting that binding may involve a degree of induced fit. It also induced structural rearrangements in the IRES that reduced RNase V1 cleavage at several sites flanking the initiation codon, in the region between domain L and a proposed irregular stem-loop structure (14) encompassing nt 854 to 902 (Fig. 1). These changes are probably due to a reduction in secondary structure in this region. Toe printing of 48S complexes indicated that the leading edge of a bound 40S subunit is at G849, in the immediate vicinity of the induced conformational changes (Fig. 7, lane 7). We therefore suggest that the eIF4G/eIF4A complex prepares a binding site for the ribosome on the IRES by inducing concerted conformational changes that include a reduction in the secondary structure of sequences centered on the initiation codon so that this region becomes accessible to an incoming 43S ribosomal complex.
The eIF4G/eIF4A complex also enhanced existing weak stops and induced new toe prints in an ATP-dependent manner downstream of the initiation codon, primarily at and in the vicinity of nt 858. These new toe prints indicate that the stability of this region had increased, which could be due to induced and possibly long-range conformational rearrangements involving sequences in this region and upstream sequences that had become unpaired by the helicase activity of the eIF4A/eIF4G complex. The possibility that sequences in the vicinity of nt 858 switch between two alternative conformations that involve similar degrees of base pairing could account for the minor changes in sensitivity of this region to chemical and enzymatic probes. Alternatively, the increased stability of this region could be due to binding of the eIF4A and/or the eIF4GI737-1116 fragment (which contains a binding site for one molecule of eIF4A). Such interactions could involve additional molecules of one or both of these factors, or could involve a single eIF4G/eIF4A complex anchored on the J-K domain that makes additional interactions with downstream sequences, which would be possible if the active center of this complex that is involved in its helicase activity is distinct from the RNA-binding surface that interacts with the J-K domain.
We are currently unable to distinguish between these two possible explanations for the enhanced stability of sequences in the vicinity of nt 858, which, we note, are not mutually exclusive. Either possibility would imply that the region between nt 850 to 900 plays a specific role in IRES function; there is evidence for this and, more specifically, for both genetic and functional interactions between this domain and the J-K domain (14, 20). A substitution in this domain suppressed the defect caused by a substitution in the A-rich loop between the J and K domains (14), and the translation defect caused by a 1-nt insertion in this loop became apparent only if sequences including the domain from nt 850 to 900 were deleted (20). The position of this stabilized domain at the 3' border of the sequence covered by a 40S subunit bound to the initiation codon AUG834 could constrain the site of ribosomal attachment and thus play a role in enhancing the accuracy of initiation codon selection.
In summary, we suggest that the eIF4G/eIF4A complex induces concerted conformational changes in the IRESs that prepare a site on the IRES to which the ribosome can bind efficiently and accurately. A requirement for these conformational changes for initiation on the IRES can account for the dependence of EMCV translation initiation on ATP (35) and on an enzymatically active eIF4G/eIF4A complex (33, 47). The basis for these requirements has until now been obscure. Thus, in addition to enhancing the affinity of eIF4G for the EMCV IRES (26) and for eIF3, a major component of the 43S complex (23), eIF4A also induces local changes in IRES conformation that prepare the region flanking and downstream of the initiation codon AUG834 so that the 43S complexes can bind to it productively, leading to initiation of translation.
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ACKNOWLEDGMENTS |
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V.G.K. and I.B.L. contributed equally to this work.
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FOOTNOTES |
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REFERENCES |
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