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Bypassing of stems versus linear base-by-base inspection of mammalian mRNAs during ribosomal scanning

2010; Springer Nature; Volume: 30; Issue: 1 Linguagem: Inglês

10.1038/emboj.2010.302

1460-2075

Irina S. Abaeva, Assen Marintchev, Vera P. Pisareva, Christopher U.T. Hellen, Tatyana V. Pestova,

RNA modifications and cancer

Article26 November 2010free access Bypassing of stems versus linear base-by-base inspection of mammalian mRNAs during ribosomal scanning Irina S Abaeva Irina S Abaeva Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Assen Marintchev Assen Marintchev Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Vera P Pisareva Vera P Pisareva Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Christopher U T Hellen Christopher U T Hellen Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Tatyana V Pestova Corresponding Author Tatyana V Pestova Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Irina S Abaeva Irina S Abaeva Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Assen Marintchev Assen Marintchev Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Vera P Pisareva Vera P Pisareva Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Christopher U T Hellen Christopher U T Hellen Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Tatyana V Pestova Corresponding Author Tatyana V Pestova Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA Search for more papers by this author Author Information Irina S Abaeva1, Assen Marintchev2, Vera P Pisareva1, Christopher U T Hellen1 and Tatyana V Pestova 1 1Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY, USA 2Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA, USA *Corresponding author. Department of Cell Biology, SUNY Downstate Medical Center, 450 Clarkson Avenue, Box 44, Brooklyn, NY 11203, USA. Tel.: +1 718 221 6121; Fax: +1 718 270 2656; E-mail: [email protected] The EMBO Journal (2011)30:115-129https://doi.org/10.1038/emboj.2010.302 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Initiation codon selection in eukaryotes involves base-by-base inspection of the 5′-untranslated region of mRNA by scanning ribosomal 43S preinitiation complexes. We employed in vitro reconstitution to investigate factor requirements for this process and report that in the absence of eIF1 and DHX29, eIFs 4A, 4B and 4G promote efficient bypassing of stable stems by scanning 43S complexes and formation of 48S initiation complexes on AUG codons immediately upstream and downstream of such stems, without their unwinding. However, intact stems are not threaded through the entire mRNA Exit channel of the 40S subunit, resulting in incorrect positioning of mRNA upstream of the ribosomal P site in 48S complexes formed on AUG codons following intact stems, which renders them susceptible to dissociation by eIF1. In 48S complexes formed on AUG codons preceding intact stems, the stems are accommodated in the A site. Such aberrant complexes are destabilized by DHX29, which also ensures that mRNA enters the mRNA-binding cleft in a single-stranded form and therefore undergoes base-by-base inspection during scanning. Introduction On most eukaryotic mRNAs, translation initiation occurs by the scanning mechanism (Jackson et al, 2010). The first step is assembly of a 43S preinitiation complex comprising a 40S ribosomal subunit, eIF2/GTP/Met-tRNAiMet, eIF3, eIF1 and eIF1A. 43S complexes attach to the 5′-proximal region of mRNA and scan to the initiation codon where they form 48S initiation complexes with established codon–anticodon base pairing. Although 43S complexes alone can bind to the 5′-end of an unstructured 5′-UTR and scan to the initiation codon, revealing their intrinsic ability to move along mRNA, ribosomal attachment and scanning on structured 5′-UTRs requires eIFs 4A, 4B and 4F, factors associated with RNA unwinding (Pestova and Kolupaeva, 2002). eIF4F comprises eIF4E (a cap-binding protein), eIF4A (a DEAD-box RNA helicase) and eIF4G (a scaffold for eIF4E and eIF4A, which also binds eIF3). The weak helicase activity of eIF4A is enhanced by eIF4G and eIF4B: eIF4G acts by aligning the DEAD-box motifs of eIF4A in a productive conformation (Schütz et al, 2008), whereas eIF4B might prevent mRNA reannealing and promote unidirectional eIF4A movement (Marintchev et al, 2009). The mRNA path on bacterial 30S subunits (Yusupova et al, 2001) comprises three regions: the Entry channel (∼12 nts), the exposed interface surface (the A, P and E sites), and the Exit channel (∼12 nts). These regions are conserved between bacterial and eukaryotic small ribosomal subunits, and the mRNA path on the 40S subunit is very similar to that in bacteria (Pisarev et al, 2008). mRNA enters the 40S subunit between the head and the shoulder and passes through a layer of ribosomal proteins (rp) including rpS2 and rpS3 and then through a layer of rRNA including helices (h) 18 in the body and 34 in the neck. h18 and h34 form a latch that is closed in free 40S subunits but opens upon binding of eIF1 and eIF1A, which occupy the areas of P and A sites, respectively (Lomakin et al, 2003; Passmore et al, 2007; Yu et al, 2009). Before exiting between the head and the platform, mRNA passes through another tunnel formed by elements that include rpS5 and h23, whereas further upstream, it is positioned close to rps S14/S26/S28 and to the 3′-end of 18S rRNA. It is not known if eIFs 4A/4G/4B act at the 40S subunit's leading edge by unwinding mRNA before it enters the mRNA-binding cleft, or if they bind near its trailing edge and assist scanning by 'pulling' mRNA through the 40S subunit. Although eIFs 4A/4B/4G can mediate scanning through stems of ΔG⩽−13.1 kcal/mol, movement of mammalian 40S subunits through stems of ΔG>−19 kcal/mol requires an additional DExH-box protein, DHX29 (Pisareva et al, 2008). Silencing of DHX29 impairs translation, resulting in polysome disassembly and accumulation of mRNA-free 80S ribosomes (Parsyan et al, 2009). DHX29 acts synergistically with eIFs 4A/4G/4B, and binds directly to 40S subunits, likely at the mRNA entrance (Pisareva et al, 2008). The mechanism by which DHX29 assists scanning is also unknown. Efficient scanning also depends on adoption by 40S subunits of a scanning-competent conformation induced by eIF1 and eIF1A: omission of eIF1A reduces the intrinsic scanning ability of 43S complexes, whereas omission of eIF1 almost abrogates it (Pestova and Kolupaeva, 2002). eIF1 also has a key role in maintaining the fidelity of initiation codon selection, enabling 43S complexes to discriminate against non-AUG triplets, and AUG triplets in suboptimal context or located within 8 nts of the 5′-end (Pestova and Kolupaeva, 2002; Lomakin et al, 2006; Pisarev et al, 2006). In a current model, eIF1 acts by antagonizing conformational changes that occur upon codon–anticodon base pairing and switch ribosomal complexes from 'open' (scanning competent) to 'closed' conformations (Lorsch and Dever, 2010). The yeast DEAD-box helicase Ded1 and its mammalian homologue DDX3 have also been implicated in initiation (Tarn and Chang, 2009). Mutational inactivation of Ded1 severely reduces polysomes and leads to accumulation of 80S monosomes (Chuang et al, 1997; de la Cruz et al, 1997). Ded1 is likely a more potent helicase than eIF4A (Marsden et al, 2006), and their functions are not redundant (Chuang et al, 1997; de la Cruz et al, 1997). This has led to a suggestion that eIF4A functions in 43S complex attachment to mRNA, whereas Ded1 promotes scanning (Marsden et al, 2006). In contrast, DDX3's involvement in initiation is controversial. Although some reports indicated that depletion of DDX3 inhibited general translation (Lee et al, 2008) or specifically affected translation of mRNAs with long or structured 5′-UTRs (Lai et al, 2008), others show that protein synthesis in a cell line expressing a ts DDX3 mutant was normal at non-permissive temperatures (Fukumura et al, 2003), and that silencing DDX3 even enhanced general translation (Shih et al, 2008). Although eIF4A, DHX29 and Ded1 have convincingly been implicated in initiation, their mechanisms of action are largely unknown. Moreover, many mechanistic aspects of scanning are also poorly understood. Thus, it is not clear what happens when a scanning 43S complex encounters a stable stem: can an intact stem at least to some extent penetrate into the mRNA-binding cleft or is it stopped at its entry, and do changes to the structure of the mRNA-binding channel induced by eIF1, eIF1A and presumably DHX29 influence mechanistic aspects of movement of structured mRNAs through the mRNA-binding cleft of the 40S subunit? Using an in vitro reconstitution approach, we investigated 48S complex formation on mRNAs containing stable stems and AUG triplets at different positions relative to them. We report that in the absence of DHX29 and eIF1, eIFs 4A/4B/4G promote efficient bypassing of stable stems by scanning 43S complexes, and formation of 48S complexes on AUGs immediately upstream and downstream of such stems without their unwinding. However, the intact stems likely cannot be threaded through the entire Exit channel, resulting in incorrect positioning of mRNA upstream of the P site that renders 48S complexes formed on AUGs downstream of intact stems susceptible to dissociation by eIF1. In 48S complexes formed on AUGs preceding stems, the stem and an adjacent mRNA region between the stem and the AUG are accommodated in the A site. Such complexes are destabilized by DHX29, which also ensures that structured mRNAs enter the mRNA-binding cleft in a single-stranded form and undergo linear base-by-base inspection during scanning. We also found that Ded1 promoted scanning on 5′-UTRs containing moderately stable stems and could act cooperatively with eIFs 4A/4B/4G. However, the activity of eIFs 4A/4B/4G in promoting scanning through more stable stems was higher when they were combined with DHX29 than with Ded1. No translational activity of DDX3 was observed in any circumstances. Results To investigate the individual and synergistic activities of eIFs 4A/4B/4G, Ded1, DDX3 and DHX29 in promoting scanning and base-by-base inspection of structured 5′-UTRs by 43S complexes, we determined their ability to mediate 48S complex formation on mRNAs containing defined stems of various stabilities and AUG codons at different positions relative to them, using a mammalian in vitro reconstituted translation system. To assess the influence of the conformation of the mRNA-binding cleft, experiments were done with and without eIF1 and eIF1A. In these studies we used native DHX29, recombinant Ded1 and DDX3 (Figure 1A) that were biochemically active and possessed RNA-dependent ATPase activity (data not shown), native eIF4F and recombinant eIF4A, eIF4B and N-terminally truncated ΔeIF4G682–1599, which we refer to as eIF4G below. Assembly of 48S complexes was monitored by the appearance of characteristic toe-prints +15–17 nts downstream of the initiation codon. Figure 1.Comparison of the activities of eIFs 4A/4B/4F, DHX29, Ded1 and DDX3 in 48S complex formation on β-globin mRNA. (A) Purified recombinant Ded1 and DDX3 resolved by SDS–PAGE. (B, C) Toe-printing analysis of 48S complex formation on native β-globin mRNA in the presence of indicated combinations of factors. Positions of the initiation codon, of the full-length cDNA and of assembled ribosomal complexes are shown on the sides of each panel. Lanes C/T/A/G depict the corresponding DNA sequences. Download figure Download PowerPoint Activities of eIFs 4A/4B/4G, DHX29, Ded1 and DDX3 in 48S complex formation on β-globin mRNA We first investigated 48S complex formation on native capped β-globin mRNA, which does not have an unstructured 5′-terminal region (Lockard et al, 1986) that would allow 43S complexes to bind without prior unwinding. Ded1 and DDX3 individually or with DHX29 did not promote 48S complex formation (Figure 1B, lanes 5, 7, 9, 11 and 12) and did not influence the efficiency of 48S complex formation mediated by eIFs 4A/4B/4F (Figure 1B, lanes 3, 6, 8 and 10). The inability of Ded1 and DDX3 to promote 48S complex formation, which contrasted with the activity of eIFs 4A/4B/4F, could not be attributed eIF4F's specific affinity to the cap because the activities of eIFs 4A/4B/4G and 4A/4B/4F were similar (Figure 1B, lanes 3 and 4). Interestingly, like DHX29 (Pisareva et al, 2008), Ded1 reduced the intensity of aberrant toe-prints +8–9 nts downstream of the AUG codon, albeit less efficiently, particularly when it was added to preassembled 48S complexes (Figure 1C). DDX3 did not influence +8–9 toe-prints even at elevated concentrations (Figure 1B, lanes 8 and 10). Importantly, as β-globin mRNA does not contain an unstructured 5′-terminal region that would allow 43S complexes to attach directly, the inability of Ded1 and DDX3 to promote 48S complex formation on it might, as in the case of DHX29 (Pisareva et al, 2008), reflect their inability to promote ribosomal attachment rather than a lack of translational activity. Individual and synergistic activities of eIFs 4A/4B/4G, DHX29, Ded1 and DDX3 in ribosomal scanning To investigate the activity of eIFs 4A/4B/4G, DHX29, Ded1 and DDX3 in promoting scanning independently of a role in ribosomal attachment, we used model (CAA)n-Stem-GUS mRNAs comprising a GUS reporter and 5′-UTRs with 43 unstructured 5′-terminal nucleotides (CAA repeats) that allow helicase-independent attachment of 43S complexes (Pestova and Kolupaeva, 2002), followed by stems of different stabilities (Figures 2A and 3A). To verify that it is base-by-base inspection of mRNAs that is being monitored, AUG codons in good context (with purines at −3 and +4 positions) were inserted into the loop of the hairpins rather than downstream of them, to exclude the potential for formation of 48S complexes through bypassing of the stem by scanning 43S complexes. The absence of near-cognate codons preceding the AUG triplet also allowed the dependence of 48S complex formation on eIF1 and eIF1A to be assayed. Figure 2.Individual and synergistic activities of eIFs 4A/4B/4G, DHX29, Ded1 and DDX3 in promoting scanning through 5′-UTRs with an internal stem of moderate stability. (A–E) Toe-printing analysis of 48S complex formation on (A–D) Stem-1 and (E) low-energy Stem mRNAs in the presence of indicated combinations of factors. Initiation codons and the positions of full-length cDNAs and of assembled ribosomal complexes are shown on the sides of each panel. Lanes C/T/A/G depict corresponding DNA sequences. Download figure Download PowerPoint Figure 3.Individual and synergistic activities of eIFs 4A/4B/4G, DHX29, Ded1 and DDX3 in promoting scanning through 5′-UTRs with a highly stable internal stem. (A) Sequence of the 5′-UTR of Stem-2 mRNA. (B–D) Toe-printing analysis of 48S complex formation on (B) Stem-1 and (B–D) Stem-2 mRNAs in the presence of indicated combinations of factors. Initiation codons and the positions of full-length cDNAs and of assembled ribosomal complexes are shown on the sides of each panel. Lanes C/T/A/G depict corresponding DNA sequences. (E) Association of Ded1 (all panels) and DHX29 (left panel) with mammalian 43S complexes (left and right panels) and yeast 40S subunits (central and right panels) was assayed by sucrose density gradient (SDG) centrifugation. Gradient fractions that corresponded to ribosomal peaks were analysed by SDS–PAGE and fluorescent SYPRO staining (left and central panels) or western blotting using anti-Ded1 antibodies (right panel). Download figure Download PowerPoint First, the individual activities of eIFs 4A/4B/4G, DHX29, Ded1 and DDX3 were compared in 48S complex formation on Stem-1 mRNA containing a GC-rich stem of moderate stability (ΔG=−13.1 kcal/mol; Figure 2A). In the absence of helicases, low-level 48S complex formation occurred on the AUG and on the AUU 10 nts downstream of Stem-1 in the presence of eIF1 alone, which was reduced further when eIF1 was combined with eIF1A (Figure 2A, lanes 2 and 4). No 48S complexes formed without eIF1 (Figure 2A, lanes 3 and 5). eIFs 4A/4B/4G promoted 48S complex formation efficiently on the AUG in the presence of eIF1 and moderately in the presence of eIF1A or eIF1/1A (Figure 2A, lanes 6–8). Surprisingly, in the presence of eIF1A alone, eIFs 4A/4B/4G also mediated high-level 48S complex formation on the AUU and even moderately on the CUG further downstream (Figure 2A, lane 7). Thus, in the presence of eIF1A, eIFs 4A/4B/4G did not ensure efficient inspection of Stem-1 by scanning 43S complexes, but instead promoted 48S complex formation downstream of the stem. In the presence of eIF1 and eIF1A together (but not individually) Ded1-mediated 48S complex formation on the AUG at a level similar to that observed in the presence of eIFs 4A/4B/4G (Figure 2A, lanes 9–11), whereas DHX29 stimulated 48S complex formation on the AUG most strongly in the presence of eIF1 alone, moderately in the presence of eIF1A alone, and weakly in the presence of both factors (Figure 2A, lanes 12–14). Interestingly, Ded1 also allowed more efficient 48S complex formation on the AUU in the presence of both eIF1 and eIF1A than eIFs 4A/4B/4G (Figure 2A, lanes 8 and 11). Moreover, initiation on the first AUG codon of mRNAs containing unstructured 5′-UTRs with two AUG triplets was considerably more efficient in the presence of Ded1 than of eIFs 4A/4B/4G, irrespective of the nucleotide context of the first AUG triplet (Supplementary Figure S1). Thus, Ded1-mediated scanning was less discriminating with respect to the context/sequence of the initiation codon than scanning mediated by eIFs 4A/4B/4G, and thus less leaky. Importantly, neither Ded1 nor DHX29 promoted efficient 48S complex formation downstream of Stem-1 in the presence of eIF1A alone (Figure 2A, lanes 10 and 13). eIFs 4A/4B/4G, Ded1 or DHX29 did not stimulate 48S complex formation when both eIF1 and eIF1A were absent (data not shown). DDX3 was not active in any circumstances (Figure 2B). We next assayed the ability of DHX29, Ded1 and DDX3 to act synergistically with eIFs 4A/4B/4G. eIFs 4A/4B/4G and DHX29 synergistically stimulated 48S complex formation on the AUG when eIF1 or eIF1A was present individually, whereas synergy between eIFs 4A/4B/4G and Ded1 required both factors (Figure 2C, lanes 6–8, 10–12). As no synergy between eIFs 4A/4B/4G and DHX29 occurred in the presence of eIF1 and eIF1A, the activity of eIFs 4A/4B/4G was higher in combination with Ded1 than with DHX29 when both eIF1 and eIF1A were present (Figure 2C, lanes 8 and 12). As in the case of individual helicases, combinations of them also did not promote 48S complex formation when both eIF1 and eIF1A were absent (Figure 2C, lanes 9 and 13). With both combination of helicases, efficient 48S complex formation on AUU and CUG triplets downstream of Stem-1 again occurred in the presence of eIF1A alone (Figure 2C, lanes 3, 7 and 11), although enhanced 48S complex assembly on the AUG in the presence of DHX29 was accompanied by a moderate reduction in 48S complex formation downstream of the stem (Figure 2C, lane 7). Importantly, elimination of GC pairs at the base of Stem-1 resulted in efficient recognition of the AUG in the loop of the stem irrespective of the presence of DHX29 and consequently, in abrogation of 48S complex formation on downstream AUU and CUG codons (Figure 2D). This confirmed that efficient recognition of the downstream AUU on Stem-1 mRNA in the presence of eIF1A alone was due to a blockage of linear scanning and skipping of the AUG in the hairpin by 43S complexes. DDX3 did not influence the activity of eIFs 4A/4B/4G in any circumstances (Figure 2E). To compare the relative strength of helicases individually and in combination, 48S complex formation was assayed on Stem-2 mRNA containing a more stable stem (ΔG=−18.9 kcal/mol) (Figure 3A). If both eIF1 and eIF1A were present, eIFs 4A/4B/4G, Ded1 and DHX29 could individually promote 48S complex formation on the AUG of Stem-1 mRNA, albeit with different efficiencies (Figure 3B, lanes 2–4), but not on Stem-2 mRNA: this required eIFs 4A/4B/4G to be combined with DHX29 or Ded1 (Figure 3B, lanes 8–12). As expected, this process strictly required eIF4G and was stimulated by eIF4B (Figure 3D, lanes 6–8). eIFs 4A/4B/4G and DHX29 functioned synergistically whether eIF1 and eIF1A were present individually or together (Figure 3C, lanes 6–8), but synergy between eIFs 4A/4B/4G and Ded1 again required both factors (Figure 3C, lanes 10–12). Interestingly, the activity of eIFs 4A/4B/4G alone and with DHX29 in promoting 48S complex formation on both Stem-1 and Stem-2 mRNAs was higher when eIF1 was present alone than with eIF1A (Figures 2B and D and 3C, lanes 6 and 8). The most likely explanation for this is that together, eIF1 and eIF1A sense deviations in the context of this AUG at positions other than the −3 and +4 consensus purines, and thus do not permit high-level 48S complex formation on it. Notably, in the presence of eIF1 and eIF1A, eIFs 4A/4B/4G were more active in 48S complex formation on Stem-2 mRNA with DHX29 than with Ded1 (Figure 3C, lanes 8 and 12), whereas the situation on Stem-1 mRNA was reversed (Figure 2C). Initiation on the AUG codon in the loop of a stem would require unwinding of the stem as well as successful recognition of the AUG codon by the scanning complex. A possible explanation for the difference in the activities of Ded1 and DHX29 in promoting initiation on the AUGs of Stem-1 and Stem-2 mRNAs could be that Stem-1 can be efficiently unwound by both sets of factors but the AUG is better recognized in the presence of Ded1 (which would be consistent with the reduced leaky scanning during Ded1-mediated 48S complex formation described above), whereas more efficient unwinding of Stem-2 can be achieved in the presence of DHX29. The step that limits initiation on the internal AUG of Stem-1 mRNA would therefore be initiation codon recognition, whereas initiation on the more stable Stem-2 mRNA would be limited by unwinding of the hairpin. Importantly, efficient eIF4A/4B/4G-mediated 48S complex formation on AUU and CUG triplets downstream of Stem-2 again occurred in the presence of eIF1A alone (Figure 3C, lanes 3, 7 and 11). There was no synergy between Ded1 and DHX29 (Figure 3D, lanes 4, 5 and 9), and DDX3 did not influence the activity of eIFs 4A/4B/4G in any circumstances (Figure 3C, lane 14; data not shown). Although Ded1 and DHX29 showed some functional similarities, unlike DHX29, Ded1 did not bind stably to mammalian 43S complexes or to yeast 40S subunits, and preincubation of 43S complexes with Ded1 did not influence their association with DHX29 (Figure 3E). This might indicate a difference in the mechanisms of coupling of the helicase activities of Ded1 and DHX29 with ribosomal complexes. In conclusion, in the presence of eIF1 and eIF1A, Ded1 (but not DDX3) was able to promote scanning on mRNAs containing moderately stable GC-rich stems and could act synergistically with eIFs 4A/4B/4G. However, eIFs 4A/4B/4G promoted scanning through more stable stems more efficiently with DHX29 than with Ded1. Importantly, in the presence of eIF1A alone, eIFs 4A/4B/4G did not mediate efficient inspection of stems by scanning 43S complexes, but instead promoted high-level 48S complex formation on downstream near-cognate codons. Bypassing of stable stems by scanning 43S complexes The efficient 48S complex formation mediated by eIFs 4A/4B/4G in the presence of eIF1A on near-cognate codons downstream of stable stems without their base-by-base inspection poses two important questions: (i) can formation of such complexes tolerate eIF1 if a near-cognate codon is replaced by an AUG triplet and (ii) do they form by 5′-end-dependent scanning or by internal ribosomal entry? To address the first question, 48S complex formation was assayed on mRNA in which the AUG in Stem-2 was eliminated, and the AUU downstream of the stem was replaced by AUG (Figure 4A, upper panel). Efficient 48S complex formation on the new AUG again occurred only in the absence of eIF1 (Figure 4A, lanes 3 and 4). As eIFs 4A/4B/4G did not promote inspection of this stem (Figure 3C), it likely remained intact in 48S complexes assembled on the downstream codon. The proximity (10 nts) of the stem to the AUG could affect correct positioning of mRNA in the Exit portion of the mRNA-binding cleft, and thus result in sensitivity of 48S complexes to dissociation by eIF1 (Pestova and Kolupaeva, 2002). However, increasing the separation between the stem and the AUG to 21 or 30 nts did not eliminate the sensitivity of 48S complex formation to eIF1 (Figure 4B and C). Moreover, even delayed addition of eIF1 to preassembled 48S complexes led to their dissociation (Figure 4B and C, lane 4). On the other hand, inclusion of DHX29 substantially enhanced 48S complex formation in eIF1's presence (Figure 4B and C, compare lanes 3, 4 with 5). Figure 4.48S complex formation on mRNAs with AUG triplets downstream of stable stems depending on the presence of eIF1 and DHX29. Toe-printing analysis of 48S complex formation on (A–C) mRNAs-containing AUG codons 10, 21 and 30 nucleotides downstream of Stem-2, respectively, (D) mRNA-containing AUG triplets 15 nts downstream of the 5′-end of mRNA, in the loop of Stem-2, and 10 nts downstream of the stem, and (E) mRNA containing an additional GC-rich stem at the extreme 5′-end and two AUG triplets, in the loop of Stem-2 and 10 nts downstream from the stem, respectively, in the presence of indicated combinations of factors. Initiation codons and the positions of full-length cDNAs and of assembled ribosomal complexes are shown on the sides of each panel. Lanes C/T/A/G depict corresponding DNA sequences. Download figure Download PowerPoint Apparently, mRNA is not properly fixed in the mRNA-binding cleft of 48S complexes formed downstream of the stem if it is not unwound, rendering them susceptible to dissociation by eIF1. To investigate if these complexes nonetheless form by scanning, two further mRNAs were used. Both contained AUGs in Stem-2 and 10 nts downstream of the stem, but the first also contained an AUG 15 nts from the 5′-end (Figure 4D, upper panel), whereas the second had a stable 5′-terminal stem (Figure 4E, upper panel). If 48S complex formation on the AUG downstream of the stem occurs by 5′-end-dependent scanning, introduction of an upstream AUG or a 5′-terminal stem should abrogate initiation on it. No 48S complexes formed on the AUG downstream of the stem on either mRNA (Figure 4D and E), confirming that 48S complex assembly on the downstream AUG occurs by 5′-end-dependent scanning. These data suggest that eIFs 4A/4B/4G enable scanning 43S complexes to bypass stable stems and to form 48S complexes downstream of them, but that intact stems likely cannot be threaded through the entire Exit portion of the mRNA-binding cleft, resulting in incorrect ribosomal positioning of mRNA upstream of the P site and consequent susceptibility of 48S complexes to dissociation by eIF1. DHX29, on the other hand, enables 43S complexes to penetrate into the stem (Figure 3C), so that in its presence, 48S complexes form on initiation codons downstream of the stem following its unwinding, and the correct positioning of unwound mRNA renders them resistant to dissociation by eIF1. Linear base-by-base inspection of mRNA by scanning 43S complexes The data described above suggest that in the absence eIF1, eIFs 4A/4B/4G enable scanning 43S complexes to bypass stable stems and to form 48S complexes downstream of them. This, in turn, raises the following questions (i) does eIF1 prevent intact stems from entering the mRNA-binding cleft or does it permit entry, but acts only later, while codon–anticodon base pairing is being established and (ii) if eIF1 does not prevent intact stems from entering the mRNA-binding cleft, then what is the mechanism that ensures that structured mRNAs enter the mRNA-binding cleft in a single-stranded form and undergo linear base-by-base inspection? To address these questions, we used an mRNA in which an AUG at position −6 relative to Stem-2 was introduced in addition to AUGs in the stem and 21 nts downstream of it (Figure 5A). We hypothesized that if an intact stem can be stably accommodated in the Entrance portion of the mRNA-binding cleft downstream of the P site, then 48S complexes formed on the AUG preceding the stem should yield toe-prints not +15–17 nts downstream of this codon, but instead a few nts downstream of the stem. Consistent with our hypothesis, eIFs 4A/4B/4G promoted efficient 48S complex formation on the AUG preceding the stem yielding toe-prints +11–12 nts downstream of the stem (with additional minor stops at +13–15 nts) (Figure 5B, lanes 2–4), and only a very small proportion of complexes with conventional +16–17 nts toe-prints assembled in the presence of both eIF1 and eIF1A (Figure 5B, lane 4). The toe-prints +11–12 nts downstream of the stem were caused by bound 40S subunits and did not appear in their absence (Figure 5B, lane 8). A small amount of 48S complexes with toe-prints +11–12 nts downstream of