The fidelity of translation initiation: reciprocal activities of eIF1, IF3 and YciH
2005; Springer Nature; Volume: 25; Issue: 1 Linguagem: Inglês
10.1038/sj.emboj.7600904
ISSN1460-2075
AutoresIvan B. Lomakin, Nikolay E. Shirokikh, Marat Yusupov, Christopher U.T. Hellen, Tatyana V. Pestova,
Tópico(s)Genomics and Phylogenetic Studies
ResumoArticle15 December 2005free access The fidelity of translation initiation: reciprocal activities of eIF1, IF3 and YciH Ivan B Lomakin Ivan B Lomakin Department of Microbiology and Immunology, SUNY Downstate Medical Center, NY, USA Search for more papers by this author Nikolay E Shirokikh Nikolay E Shirokikh Department of Microbiology and Immunology, SUNY Downstate Medical Center, NY, USA Search for more papers by this author Marat M Yusupov Marat M Yusupov Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France Search for more papers by this author Christopher UT Hellen Christopher UT Hellen Department of Microbiology and Immunology, SUNY Downstate Medical Center, NY, USA Search for more papers by this author Tatyana V Pestova Corresponding Author Tatyana V Pestova Department of Microbiology and Immunology, SUNY Downstate Medical Center, NY, USA AN Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Ivan B Lomakin Ivan B Lomakin Department of Microbiology and Immunology, SUNY Downstate Medical Center, NY, USA Search for more papers by this author Nikolay E Shirokikh Nikolay E Shirokikh Department of Microbiology and Immunology, SUNY Downstate Medical Center, NY, USA Search for more papers by this author Marat M Yusupov Marat M Yusupov Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France Search for more papers by this author Christopher UT Hellen Christopher UT Hellen Department of Microbiology and Immunology, SUNY Downstate Medical Center, NY, USA Search for more papers by this author Tatyana V Pestova Corresponding Author Tatyana V Pestova Department of Microbiology and Immunology, SUNY Downstate Medical Center, NY, USA AN Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Author Information Ivan B Lomakin1,‡, Nikolay E Shirokikh1,‡, Marat M Yusupov2,3, Christopher UT Hellen1 and Tatyana V Pestova 1,4 1Department of Microbiology and Immunology, SUNY Downstate Medical Center, NY, USA 2Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France 3Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France 4AN Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia ‡These authors contributed equally to this work *Corresponding author. Department of Microbiology and Immunology, 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 (2006)25:196-210https://doi.org/10.1038/sj.emboj.7600904 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Eukaryotic initiation factor eIF1 and the functional C-terminal domain of prokaryotic initiation factor IF3 maintain the fidelity of initiation codon selection in eukaryotes and prokaryotes, respectively, and bind to the same regions of small ribosomal subunits, between the platform and initiator tRNA. Here we report that these nonhomologous factors can bind to the same regions of heterologous subunits and perform their functions in heterologous systems in a reciprocal manner, discriminating against the formation of initiation complexes containing codon–anticodon mismatches. We also show that like IF3, eIF1 can influence initiator tRNA selection, which occurs at the stage of ribosomal subunit joining after eIF5-induced hydrolysis of eIF2-bound GTP. The mechanisms of initiation codon and initiator tRNA selection in prokaryotes and eukaryotes are therefore unexpectedly conserved and likely involve related conformational changes induced in the small ribosomal subunit by factor binding. YciH, a prokaryotic eIF1 homologue, could perform some of IF3's functions, which justifies the possibility that YciH and eIF1 might have a common evolutionary origin as initiation factors, and that IF3 functionally replaced YciH in prokaryotes. Introduction The fidelity of translation depends on accurate selection of the correct reading frame during initiation. In eukaryotes, this process involves at least 11 eukaryotic initiation factors (eIFs). Met-tRNAiMet forms a ternary complex with eIF2 and GTP, which together with eIF1, eIF1A and eIF3 binds to the 40S ribosomal subunit to form a 43S preinitiation complex. After loading onto the mRNA's 5′ end in a process requiring eIFs 4A, 4B and 4F, the 43S complex scans downstream until it encounters an AUG triplet in a favorable context GCC(A/G)CCAUGG (in which the nucleotides at the −3 and +4 positions are the most important; Kozak, 1991), stops and forms a stable 48S complex with established codon–anticodon base pairing in the P site. These two context nucleotides are important features of mammalian mRNAs but differ in sequence and importance in other eukaryotes. Subsequent joining of a 60S subunit is mediated by eIF5 (which induces hydrolysis of eIF2-bound GTP and dissociation of eIF2-GDP from the 40S subunit) and eIF5B (which mediates subunit joining and dissociation of other factors) (Pestova et al, 2000; Unbehaun et al, 2004). eIF1 plays the key role in initiation codon selection. In an in vitro reconstituted system, eIF1 enables scanning 43S complexes to discriminate against non-AUG triplets and AUG triplets that have poor context or are located within 4 nt. of the 5′ end of an mRNA, and also promotes dissociation of ribosomal complexes aberrantly assembled at such triplets in its absence (Pestova et al, 1998; Pestova and Kolupaeva, 2002). Mutations in eIF1 permit initiation at noncognate initiation codons in vivo in yeast (e.g. Yoon and Donahue, 1992). eIF1 also prevents premature eIF5-induced hydrolysis of eIF2-bound GTP before codon–anticodon base pairing has been established (Unbehaun et al, 2004). By contrast, initiation in prokaryotes requires only three initiation factors (IFs), does not involve scanning, and the 30S ribosomal subunit itself plays a direct role in initiation codon selection, by binding to the Shine–Dalgarno sequence upstream of the initiation codon (Laursen et al, 2005). IF3 increases the accuracy of initiation codon selection by promoting dissociation of pseudo-initiation complexes assembled either on noninitiation codons, or with noninitiator tRNA, particularly discriminating against mutations in three conserved G-C pairs in its anticodon stem, and also modulates the translation efficiency of leaderless mRNAs (Hartz et al, 1990; Meinnel et al, 1999; Dallas and Noller, 2001; Petrelli et al, 2001). IF3's proofreading function requires only its C-terminal domain (IF3-CTD) (Petrelli et al, 2001). IF3 thus displays several activities that resemble those of eIF1 in ensuring the fidelity of initiation. Moreover, both eIF1 and IF3-CTD bind to the same region of the small ribosomal subunit, between the platform and initiator tRNA (Dallas and Noller, 2001; Lomakin et al, 2003). This location corresponds to a region of high homology between 16S and 18S rRNA. These observations suggest that eIF1 and IF3 may influence initiation codon selection by similar mechanisms. Ribosome-bound eIF1 and IF3-CTD are both out of reach of the initiation codon and the conserved G-C base pairs in the anticodon stem of initiator tRNA, so that any role for these factors in promoting the fidelity of initiation must be indirect. Binding of IF3 induces changes in the conformation of the 30S subunit and in its interaction with mRNA (Shapkina et al, 2000; Petrelli et al, 2001). It has been proposed that IF3-induced tilting of the 30S subunit head toward the platform and juxtaposition of GA1338–9 of 16S rRNA with the anticodon stem of initiator tRNA could constitute a means for checking the identity of tRNA in the P site (Dallas and Noller, 2001). Despite similarities in function, size, shape and charge distribution (Lomakin et al, 2003), there is no sequence or structural homology between eIF1 and IF3-CTD. This is surprising in light of the homology between other prokaryotic and eukaryotic initiation factors, and between the regions of the small ribosomal subunit to which they both bind. However, all archaea and some prokaryotes encode homologues of eIF1 (Kyrpides and Woese, 1998), which in enteric bacteria is known as YciH (Cort et al, 1999). YciH is nonessential and its function is unknown. These similarities between eIF1 and IF3 prompted us to test if they could perform some functions in heterologous systems in a reciprocal manner. We now report that eIF1 and IF3 bind to identical regions on homologous and heterologous ribosomal subunits, and can discriminate against the formation of initiation complexes with codon–anticodon mismatches in heterologous systems. Moreover, like IF3, eIF1 can influence initiator tRNA selection, but this activity is manifested only after hydrolysis of eIF2-bound GTP and dissociation of eIF2-GDP from initiator tRNA in the P site. Related conformational changes induced in the small ribosomal subunit may therefore be responsible for the selection of the initiation codon and initiator tRNA by eIF1 and IF3. The mechanisms of initiation codon and initiator tRNA selection in prokaryotes and eukaryotes are therefore unexpectedly conserved. YciH was able to perform some of the functions of IF3 in prokaryotic initiation, an observation that justifies the possibility that YciH and eIF1 have a common evolutionary origin as initiation factors, and that IF3 has functionally replaced YciH in prokaryotes. Results Binding of eIF1, IF3 and YciH to 40S and 30S ribosomal subunits Although eIF1 and IF3-CTD have unrelated sequences and structures, their functions in ensuring the fidelity of initiation codon selection and positions on the small ribosomal subunit are similar. The homology of their respective binding sites on eukaryotic 40S and prokaryotic 30S subunits (Spahn et al, 2001) prompted us to test their binding to heterologous subunits. YciH was included in these studies. In pull-down experiments, T7-tagged IF3 and IF3-CTD bound 40S subunits as well as eIF1, whereas binding of YciH was lower (Figure 1A). Untagged eIF1 competed with YciH but not with IF3 for binding to 40S subunits (Figure 1B), indicating that YciH and eIF1 bind to the same or overlapping sites. Our failure to detect eIF1 in lanes 3 and 4 suggested that rather than binding to different sites on 40S subunits, eIF1 and IF3 bind to the same site but IF3's affinity is higher. To confirm this, we investigated competition between eIF1 and IF3 for binding to 40S subunits using sucrose density gradient centrifugation. Although we previously noted that this method is not very suitable for studying the eIF1/40S subunit interaction because these complexes are unstable under centrifugation conditions (Lomakin et al, 2003), loading of large amounts of eIF1/40S subunit complex on sucrose gradients permitted us to detect eIF1 and IF3 in ribosomal complexes. Incubation of preformed eIF1/40S subunit complexes with IF3 reduced binding of eIF1 to 40S subunits approximately four-fold (Figure 1C), confirming that eIF1 and IF3 bind to the same or overlapping sites. In pull-down experiments, eIF1 and YciH also bound to 30S subunits (Figure 1D), and again, the ribosome binding activity of YciH was lower. Figure 1.Binding of IF3, YciH and eIF1 to (A–C) 40S subunits and (D) 30S subunits. (A, B, D) Interaction of ribosomal subunits with T7-tag antibody agarose-immobilized eIF1, IF3, IF3-CTD or YciH (as indicated), and (B) in the presence of recombinant untagged eIF1 (as indicated) in in vitro binding assays. Ribosomal proteins and initiation factors were stained with Coomassie blue. Initiation factors are indicated by red arrows. (C) Presence of T7-tagged eIF1 and IF3 in ribosomal complexes isolated from sucrose density gradients (lanes 1 and 2), and eIF1 and IF3 markers (lanes 3 and 4) visualized by Western blotting. Download figure Download PowerPoint The eIF1-binding site on the 30S subunit was determined by specific cleavage of 16S rRNA by hydroxyl radicals generated at Fe(II) site, specifically tethered to unique cysteine residues on the surface of 30S-bound eIF1, after treatment with ascorbic acid and H2O2. Hydroxyl radicals have a small radius of action (∼20 Å), which allows precise localization of eIF1. To determine its orientation on 30S subunits, we used four eIF1 mutants with single surface-exposed cysteine residues (Figure 2A) previously used to locate eIF1 on 40S subunits (Lomakin et al, 2003). Cleavage of 16S rRNA at nt. 694–696 (helix 23b) from Cys75, at nt. 1400 (helix 44) from Cys38 and Cys42, and at nt. 784–789 (helix 24a) from Cys38, Cys42, Cys61 and Cys75 (Figures 2B–H) was identical to cleavage of 18S rRNA from these residues in eIF1/40S subunit complexes (Lomakin et al, 2003). These cleavage sites in the 16S rRNA and eIF1's position on the 40S subunit were modeled onto a Thermus thermophilus 30S subunit (Supplementary Figure 1A). In addition to these cleavage sites, which are consistent with eIF1 binding to identical positions on 40S and 30S subunits, Cys61 and Cys75 also cleaved 16S rRNA at nt. 722–723 (helix 23a) and nt. 830–832 (helix 26) (Figures 2B, C, D, I and J). This second set of cleavage sites on the solvent side of the 30S subunit (Supplementary Figure 1B) would be consistent with eIF1 binding to a position like that of IF3-CTD in IF3-CTD/30S subunit complexes as determined by X-ray crystallography of IF3-CTD soaked into 30S subunit crystals (Pioletti et al, 2001). We therefore cannot exclude the possibility of two eIF1-binding sites on a 30S subunit with potentially different affinities. Figure 2.Directed hydroxyl radical cleavage of 16S rRNA in 30S/eIF1 complexes from Fe(II) tethered to different positions on eIF1. (A) Ribbon diagram of the structured domain of eIF1. Colored spheres indicate the positions of cysteines introduced to tether Fe(II)-BABE. (B) Secondary structure of Escherichia coli 16S rRNA. Sites of directed hydroxyl radical cleavage are shown as red bars. (C, E, G, I) Primer extension analysis of directed hydroxyl radical cleavage of 16S rRNA (in helices 23, 44, 24 and 26, respectively) from Fe(II) tethered to positions on eIF1 as indicated. Cleavage sites are indicated to the left of each panel. Lanes marked 'Cys-less' correspond to reaction mixtures that contained the cysteine-less eIF1 mutant. Lanes C, T, A and G depict 16S rRNA sequence generated from the same primer. (D, F, H, J) Elements of helices 23, 44, 24 and 26 of 16S rRNA with hydroxyl radical cleavage sites (red circles). Download figure Download PowerPoint As the results of pull-down and sucrose density gradient centrifugation experiments, and detection of hydroxyl radical cleavage in helices 44, 23b and 24a of 16S rRNA in eIF1/30S subunit complexes both suggest that eIF1, IF3-CTD and YciH bound to similar regions on 40S and 30S subunits, we compared their activities in eukaryotic and prokaryotic translation initiation. Activities of eIF1, IF3 and YciH in ribosomal dissociation and subunit antiassociation Unlike IF3, eIF1 alone has no ribosome dissociation or antiassociation activity, but consistent with its position on 40S subunits (which would block access of 60S subunits to 18S rRNA elements that form B2b and B2d intersubunit bridges; Spahn et al, 2001), it strongly enhances the ribosome dissociation/antiassociation activity of eIF3 (Kolupaeva et al, 2005). That IF3-CTD can dissociate 70S ribosomes while eIF1 cannot dissociate 80S ribosomes despite binding to the same area of 30S/40S subunits could be because of differences in the affinity of these factors to 30S and 40S subunits or in intersubunit interactions of prokaryotic and eukaryotic ribosomes (and thus in mechanisms of subunit association). We therefore tested the dissociation activity of eIF1 on 70S ribosomes, of IF3 on 80S ribosomes and of YciH on both. eIF1 did not dissociate 80S or 70S ribosomes and did not protect 30S subunits from association with 50S subunits (Figures 3A and B; data not shown). IF3-CTD, capable of dissociating 70S ribosomes, could not dissociate 80S ribosomes or prevent association of 40S and 60S subunits, and YciH had no dissociation/anti-association activity in either system (Figures 3A and B; data not shown). Although IF3, eIF1 and YciH bind to identical/overlapping regions of 70S and 80S ribosomes, only IF3-CTD had ribosome dissociation activity and it was specific for 70S ribosomes. This activity of IF3 in the prokaryotic system could be because only IF3-CTD binds sufficiently avidly to 30S subunits, or because the architecture of IF3-CTD/30S subunit complexes causes bound IF3 to inhibit subunit association at an early stage when it cannot be displaced by 50S subunits. Figure 3.Activities of eIF1, IF3, IF3-CTD and YciH in promoting ribosomal dissociation and formation of 43S complexes. Dissociation of (A) 70S and (B) 80S ribosomes in the presence of factors as indicated. The optical density of ribosomal profiles was measured after centrifugation through 10–30% sucrose density gradients. The positions of ribosomes and ribosomal subunits are indicated. (C, D) 43S complex formation in reaction mixtures containing [35S]Met-tRNAiMet, 40S subunits and factors as indicated was assayed by centrifugation through 10–30% sucrose density gradients. Aliquots of gradient fractions were analyzed by scintillation counting. The position of 43S complexes is indicated. Fractions from upper parts of gradients were omitted for clarity. Download figure Download PowerPoint Activities of eIF1, IF3 and YciH in 43S complex formation eIF1 stimulates binding of eIF2-ternary complexes to 40S subunits strongly in the presence of eIF1A and relatively less so in the presence of eIF3, although the overall binding of eIF2-ternary complexes to 40S subunits is higher in the presence of eIF3 (Kolupaeva et al, 2005). Neither IF3/IF3-CTD nor YciH enhanced 43S complex formation (Figures 3C and D; data not shown). The mechanism by which eIF1 stimulates this process is not known, but it is likely indirect, and IF3 and YciH may not induce the necessary conformational changes in 40S subunits. The relative activity of eIF1 in stimulating 43S complex formation in the presence of both eIF3 and eIF1A is very low (Kolupaeva et al, 2005), and the activities of IF3 and YciH were therefore not tested in this combination. Activities of IF3 and YciH in dissociating aberrant eukaryotic 48S complexes We also investigated if IF3 and YciH can play any of eIF1's roles in maintaining the fidelity of initiation codon selection in eukaryotes. Toe-printing analysis was used to test if IF3, IF3-CTD and YciH can dissociate complex I formed at the 5′-end of native capped β-globin mRNA and aberrant ribosomal complexes formed on AUG triplets located 1 nt. from the 5′-end of mRNA, on near-cognate initiation codons, or on AUG triplets in bad nucleotide context using mRNAs (Figure 4A) that are derivatives of (CAA)n-GUS mRNA containing a GUS reporter gene and an unstructured 5′-UTR lacking potential near-cognate initiation codons (Figure 6B). Figure 4.Activities of eIF1, IF3, IF3-CTD and YciH in dissociating aberrant eukaryotic 48S complexes. (A) Sequences of 5′-UTRs of β-globin mRNA and (CAA)n-GUS mRNA derivatives with initiation codons in bold. (B–F) Toe-printing analysis of 48S complexes assembled on mRNAs as indicated. Reaction mixtures contained 40S subunits, Met-tRNAiMet and factors as indicated. The positions of toe-prints caused by assembled 48S complexes are shown on the left. Full-length cDNAs are labeled. (E) 'Complex I' indicates the position of toe-prints caused by ribosomal complexes whose leading edge was 21–24 nt. from the 5′-end of β-globin mRNA. (B, C) Lanes marked C, T, A and G show cDNA sequences derived using the same primer as that for toe-printing. Download figure Download PowerPoint In the presence of eIF1, 48S complexes formed only on the GUS start codon of 1 nt.-AUG-(CAA)n-GUS mRNA, but in its absence, formed almost exclusively on the 5′-proximal AUG (Figure 4B, lanes 2 and 3; Pestova and Kolupaeva, 2002). Like eIF1, IF3 also promoted 48S complex formation predominantly on the GUS initiation codon (Figure 4B, lane 4). IF3-CTD dissociated ∼70% of 48S complexes from the 5′-proximal AUG and enhanced 48S complex formation on the GUS initiation codon accordingly, whereas YciH was less active and dissociated only ∼25% of 5′-proximal 48S complexes and formed fewer 48S complexes on the GUS initiation codon (Figure 4B, lanes 5 and 6). A stem inserted into the 5′-UTR of (CAA)n-GUS mRNA contained a near-cognate AUU initiation codon (Figure 4A). In the absence of eIF1, 48S complexes formed efficiently on this triplet (Figure 5C, lanes 2 and 3; Pestova and Kolupaeva, 2002). Like eIF1, IF3 promoted exclusive 48S complex formation on the GUS initiation codon (Figure 4C, lane 4). IF3-CTD and YciH reduced 48S complex formation on the AUU triplet by ∼90 and ∼75%, respectively (Figure 4C, lanes 5 and 6). Figure 5.Activities of eIF1, IF3 and YciH in initiation codon and initiator tRNA selection in prokaryotes. (A) The sequences of the 5′UTRs of SD-AUG and SD-AUU mRNAs. The Shine–Dalgarno sequence is bold and underlined; initiation codons are bold. (B–D) Toe-printing analysis of prokaryotic initiation complexes assembled on (B, D) SD-AUG mRNA and (C) SD-AUU mRNA. Reaction mixtures contained 30S subunits, IF1 and wild-type E.coli tRNAiMet or CCC-GGG mutant transcript tRNAiMet (Figure 6A) as well as IF3, eIF1 and YciH as indicated. Assembly reactions in (D) were carried out at 6 or 10 mM Mg2+ as indicated. Full-length cDNAs are labeled; cDNAs labeled '+16 nt. from AUG' or '+16 nt. from AUU' correspond to toe-prints caused by initiation complexes. Lanes marked G, A, T and C show cDNA sequences derived using the same primer as that for toe-printing. Download figure Download PowerPoint Discrimination of initiation codon context by IF3 and YciH was investigated using mRNA with an AUG triplet in 'bad' context upstream of the GUS initiation codon (Figure 4A). In the presence of eIF1, ∼90% of 43S complexes scanned to the GUS initiation codon, whereas in the absence of eIF1, 48S complexes assembled mostly on the first AUG triplet despite its bad context (Figure 4D, lanes 2 and 3; Pestova and Kolupaeva, 2002). IF3 also caused 48S complexes to form almost exclusively on the GUS initiation codon, whereas YciH reduced 48S complex formation on the bad context AUG by only 15% (Figure 4D, lanes 4 and 5). In the absence of eIF1, an aberrant ribosomal complex I forms at the 5′-end of native β-globin mRNA (Figure 5E, lanes 2 and 3; Pestova et al, 1998). Its formation was reduced by 60% by IF3 and by 15–20% by YciH or IF3-CTD (Figure 4E, lanes 3–6). However, in contrast to eIF1, neither IF3/IF3-CTD nor YciH promoted efficient 48S complex formation on the correct initiation codon (Figure 4E, lanes 4–6). Formation of complex I is dependent on the eIF4F–cap interaction. Thus consistent with our previous data (Pestova and Kolupaeva, 2002), complex I did not form on uncapped β-globin mRNA in the absence of eIF1, but instead small amounts of 48S complex were formed at the AUG codon of β-globin mRNA, and an additional aberrant ribosomal complex was formed at a near-cognate GUG initiation codon in the β-globin 5′-UTR (Figure 4F, lane 2). Like eIF1, IF3 promoted exclusive 48S complex formation on the initiation codon of β-globin mRNA, but with an efficiency of only half that of eIF1 (Figure 4F, lanes 3 and 4). In conclusion, IF3, IF3-CTD and YciH all discriminated against 48S complex formation on a near-cognate initiation codons, on AUG triplets at the 5′-end of mRNA or with bad context, and against formation of aberrant ribosomal complex I on β-globin mRNA. Full-length IF3 was most and YciH least active. Dissociation of prokaryotic initiation complexes assembled on AUU triplets by eIF1 and YciH We next examined the ability of eIF1 and YciH to dissociate prokaryotic initiation complexes containing codon–anticodon mismatches using mRNAs with AUG or AUU codons and canonical Shine–Dalgarno (S–D) sequences (Figure 5A). To increase initiation efficiency, these elements were flanked by multiple CAA triplets to minimize secondary structure. Binding of initiator tRNA to 30S subunits does not require IF2 (e.g. Hartz et al, 1989). This allowed us to assemble initiation complexes from only 30S subunits, mRNA, Escherichia coli tRNAiMet and IF1. Inclusion of eIF1 or YciH did not influence initiation complex formation on mRNA containing an AUG triplet, but N-terminally tagged IF3 surprisingly reduced its formation by 60% (Figure 5B, lanes 1–4). Since native untagged IF3 also reduced initiation complex formation on mRNA containing an AUG triplet (data not shown) and showed the same activities as N-terminally tagged IF3 in all assays presented in Figure 4 (data not shown), we conclude that an N-terminal tag did not alter IF3's activity. Ribosomal complexes did not form at all on SD-AUU mRNA in the presence of either IF3 or eIF1, and YciH reduced complex formation 20-fold (Figure 5C, lanes 3–6). In conclusion, like IF3, eIF1 and YciH discriminated against prokaryotic initiation complexes with codon–anticodon mismatches. Activities of eIF1, IF3 and YciH in dissociation of eukaryotic 48S complexes assembled with initiator tRNAs containing mutations in the anticodon stem Both eIF1 and IF3 monitor the fidelity of initiation codon selection but IF3 also participates in initiator tRNA selection, discriminating against initiator tRNA with mutations in three conserved GC pairs in its anticodon stem (Hartz et al, 1989, 1990). IF3 has been proposed to mediate selection indirectly by inducing conformational changes in the 30S subunit, tilting the head towards the platform and enabling GA1338–9 of 16S rRNA in the head to inspect the minor groove of the anticodon stem in the region of these GC pairs (Dallas and Noller, 2001). Eukaryotic tRNAiMet also contains these GC pairs and although it is stringently selected by eIF2, eIF1 might also contribute to selection if it induces conformational changes in 40S subunits similar to those induced in 30S subunits by IF3. To investigate this potential role of eIF1, we used tRNAiMet mutants in which three GC pairs were reversed (CCC-GGG mutant) or in which two were substituted by AU pairs (AGU-UCA mutant) (Figure 6A). Both tRNAs were as active as wt tRNAiMet transcripts or native tRNAiMet in eIF2/GTP/Met-tRNAi ternary complex and 43S complex formation (data not shown). We therefore assayed their activity in 48S complex formation with or without eIF1 on (CAA)n-GUS mRNA (Figure 6B), which has no potential near-cognate initiation codons in its 5′-UTR and which allows 48S complex assembly on the GUS initiation codon in the absence of eIF1 (Pestova and Kolupaeva, 2002). We also determined the influence on 48S complex formation of eIF5-induced hydrolysis of eIF2-bound GTP, after which tRNAiMet remains on the 40S subunit but is no longer bound to eIF2. IF3 and YciH were also assayed. In the absence of eIF5-stimulated hydrolysis of eIF2-bound GTP, eIF1, IF3 and YciH did not affect 48S complex formation on (CAA)n-GUS mRNA with wt native or in vitro transcribed tRNAiMet (Figure 6C, lanes 2, 4 and 6; Figure 6D, lanes 2–4 and 9). However, in the presence of eIF5, IF3 dissociated ∼90% of 48S complexes assembled with tRNAiMet transcripts but not with native wt tRNAiMet (Figure 6C, lane 5; Figure 6D, lane 7). Neither eIF1 nor YciH dissociated 48S complexes in the presence of eIF5, and eIF1 did not protect 48S complexes containing wt tRNAiMet transcripts from dissociation by IF3 (Figure 6D, lanes 6, 8 and 10). eIF1, IF3 or YciH did not discriminate against 48S complex formation with CCC-GGG or AGU-UCA mutant tRNAs in the absence of eIF5-induced hydrolysis of eIF2-bound GTP (Figures 6E and F, lanes 2–4), but these complexes were dissociated almost completely by IF3 and by 70% by eIF1 (Figures 6E and F, lanes 6 and 7) following hydrolysis of eIF2-bound GTP. YciH did not dissociate 48S complexes containing mutant tRNAs in the presence of eIF5 (Figures 6E and F, lane 8). The activities of eIF1, IF3 and YciH in the dissociation of initiation complexes assembled with wt or mutant in vitro transcribed initiator tRNAs did not depend on the order of mixing and incubation of components. Thus, identical results were obtained if eIF5 and eIF1, IF3 or YciH were added simultaneously with all other translational components, or if initiation complexes were first assembled without these factors, then incubated for 15 min with eIF5 to promote complete hydrolysis of eIF2-bound GTP and after that for 10 min with eIF1, IF3 or YciH (data not shown). The fact that eIF1 dissociated a large proportion of 48S complexes assembled with mutant tRNAiMet after hydrolysis of eIF2-bound GTP suggests that initiator tRNA selection occurs at two stages. Initial selection occurs during eIF2-ternary complex formation and involves only eIF2. A second selection step involves eIF1 and occurs after eIF5-stimulated hydrolysis of eIF2-bound GTP and release of eIF2/GDP from initiation complexes. eIF1 therefore ensures the fidelity of initiation codon selection during 48S complex formation and ensures the fidelity of initiator tRNA selection at the later ribosomal subunit joining stage. Activities of eIF1 and YciH in dissociating prokaryotic initiation complexes assembled wi
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