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Eukaryotic translation initiation is a multistep process requiring a number of eukaryotic translation initiation factors (eIFs). Two GTPases play key roles in the process. eIF2 brings the initiator Met-tRNA(i) to the pre-initiation complex (PIC). Upon start codon selection and GTP hydrolysis promoted by the GTPase-activating protein (GAP) eIF5, eIF2-GDP is displaced from Met-tRNA(i) by eIF5B-GTP and is released in complex with eIF5. eIF5B promotes ribosomal subunit joining, with the help of eIF1A. Upon subunit joining, eIF5B hydrolyzes GTP and is released together with eIF1A. We found that human eIF5 interacts with eIF5B and may help recruit eIF5B to the PIC. An eIF5B-binding motif was identified at the C-terminus of eIF5, similar to that found in eIF1A. Indeed, eIF5 competes with eIF1A for binding and has ~100-fold higher affinity for eIF5B. Since eIF5 is the GAP of eIF2, the newly discovered interaction offers a possible mechanism for coordination between the two steps in translation initiation controlled by GTPases: start codon selection and ribosomal subunit joining. Our results indicate that in human, eIF5B displacing eIF2 from Met-tRNA(i) upon subunit joining may be coupled to eIF1A displacing eIF5 from eIF5B, allowing the eIF5:eIF2-GDP complex to leave the ribosome.

Ribosomal attachment to mammalian capped mRNAs is achieved through the cap–eukaryotic initiation factor 4E (eIF4E)–eIF4G–eIF3–40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5′-terminal AUGs was stimulated by the eIF4E–cap interaction and followed “the first AUG” rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5′ end of mRNA, implying that Met-tRNAiMet inspects mRNA from the first nucleotide and that initiation does not have a “blind spot.” In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E–eIF4G–eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E–eIF4G–eIF3–40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAiMet can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m7G cap specifically interacts with eIF3l.

Dicistrovirus intergenic region internal ribosomal entry sites (IGR IRES) do not require initiator tRNA, an AUG codon or initiation factors, and jumpstart translation from the middle of the elongation cycle via formation of IRES/80S complexes resembling the pre-translocation state. eEF2 then translocates the [codon-anticodon]-mimicking pseudoknot I (PKI) from ribosomal A to P sites, bringing the first sense codon into the decoding center. Halastavi árva virus (HalV) contains an IGR that is related to previously described IGR IRESs, but lacks domain 2, which enables these IRESs to bind to individual 40S ribosomal subunits. By employing in vitro reconstitution and cryo-electron microscopy, we now report that the HalV IGR IRES functions by the simplest initiation mechanism that involves binding to 80S ribosomes such that PKI is placed in the P site, so that the A site contains the first codon that is directly accessible for decoding without prior eEF2-mediated translocation of PKI.

Halastavi árva virus (HalV) has a positive-sense RNA genome, with an 827 nt-long 5′ UTR and an intergenic region separating two open reading frames. Whereas the encoded proteins are most homologous to Dicistrovirus polyproteins, its 5′ UTR is distinct. Here, we report that the HalV 5′ UTR comprises small stem-loop domains separated by long single-stranded areas and a large A-rich unstructured region surrounding the initiation codon AUG828, and possesses cross-kingdom internal ribosome entry site (IRES) activity. In contrast to most viral IRESs, it does not depend on structural integrity and specific interaction of a structured element with a translational component, and is instead determined by the unstructured region flanking AUG828. eIF2, eIF3, eIF1 and eIF1A promote efficient 48S initiation complex formation at AUG828, which is reduced ∼5-fold on omission of eIF1 and eIF1A. Initiation involves direct attachment of 43S preinitiation complexes within a short window at or immediately downstream of AUG828. 40S and eIF3 are sufficient for initial binding. After attachment, 43S complexes undergo retrograde scanning, strongly dependent on eIF1 and eIF1A. eIF4A/eIF4G stimulated initiation only at low temperatures or on mutants, in which areas surrounding AUG828 had been replaced by heterologous sequences. However, they strongly promoted initiation at AUG872, yielding a proline-rich oligopeptide.

During eukaryotic translation initiation, 43S complexes, comprising a 40S ribosomal subunit, initiator transfer RNA and initiation factors (eIF) 2, 3, 1 and 1A, attach to the 5'-terminal region of messenger RNA and scan along it to the initiation codon. Scanning on structured mRNAs also requires the DExH-box protein DHX29. Mammalian eIF3 contains 13 subunits and participates in nearly all steps of translation initiation. Eight subunits having PCI (proteasome, COP9 signalosome, eIF3) or MPN (Mpr1, Pad1, amino-terminal) domains constitute the structural core of eIF3, to which five peripheral subunits are flexibly linked. Here we present a cryo-electron microscopy structure of eIF3 in the context of the DHX29-bound 43S complex, showing the PCI/MPN core at ∼6 Å resolution. It reveals the organization of the individual subunits and their interactions with components of the 43S complex. We were able to build near-complete polyalanine-level models of the eIF3 PCI/MPN core and of two peripheral subunits. The implications for understanding mRNA ribosomal attachment and scanning are discussed.

The cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the CrPV IRES jumpstarts translation in the elongation phase from the ribosomal A site. Here, we present cryoelectron microscopy (cryo-EM) maps of 80S⋅CrPV-STOP ⋅ eRF1 ⋅ eRF3 ⋅ GMPPNP and 80S⋅CrPV-STOP ⋅ eRF1 complexes, revealing a previously unseen binding state of the IRES and directly rationalizing that an eEF2-dependent translocation of the IRES is required to allow the first A-site occupation. During this unusual translocation event, the IRES undergoes a pronounced conformational change to a more stretched conformation. At the same time, our structural analysis provides information about the binding modes of eRF1 ⋅ eRF3 ⋅ GMPPNP and eRF1 in a minimal system. It shows that neither eRF3 nor ABCE1 are required for the active conformation of eRF1 at the intersection between eukaryotic termination and recycling.

Picornavirus Type 1 IRESs comprise five principal domains (dII–dVI). Whereas dV binds eIF4G, a conserved AUG in dVI was suggested to stimulate attachment of 43S ribosomal preinitiation complexes, which then scan to the initiation codon. Initiation on Type 1 IRESs also requires IRES trans-acting factors (ITAFs), and several candidates have been proposed. Here, we report the in vitro reconstitution of initiation on three Type 1 IRESs: poliovirus (PV), enterovirus 71 (EV71), and bovine enterovirus (BEV). All of them require eIF2, eIF3, eIF4A, eIF4G, eIF4B, eIF1A, and a single ITAF, poly(C) binding protein 2 (PCBP2). In each instance, initiation starts with binding of eIF4G/eIF4A. Subsequent recruitment of 43S complexes strictly requires direct interaction of their eIF3 constituent with eIF4G. The following events can differ between IRESs, depending on the stability of dVI. If it is unstructured (BEV), all ribosomes scan through dVI to the initiation codon, requiring eIF1 to bypass its AUG. If it is structured (PV, EV71), most initiation events occur without inspection of dVI, implying that its AUG does not determine ribosomal attachment.

Type 2 internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV) and other picornaviruses comprise five major domains H-L. Initiation of translation on these IRESs begins with specific binding of the central domain of initiation factor, eIF4G to the J-K domains, which is stimulated by eIF4A. eIF4G/eIF4A then restructure the region of ribosomal attachment on the IRES and promote recruitment of ribosomal 43S pre-initiation complexes. In addition to canonical translation factors, type 2 IRESs also require IRES trans-acting factors (ITAFs) that are hypothesized to stabilize the optimal IRES conformation that supports efficient ribosomal recruitment: the EMCV IRES is stimulated by pyrimidine tract binding protein (PTB), whereas the FMDV IRES requires PTB and ITAF(45). To test this hypothesis, we assessed the effect of ITAFs on the conformations of EMCV and FMDV IRESs by comparing their influence on hydroxyl radical cleavage of these IRESs from the central domain of eIF4G. The observed changes in cleavage patterns suggest that cognate ITAFs promote similar conformational changes that are consistent with adoption by the IRESs of comparable, more compact structures, in which domain J undergoes local conformational changes and is brought into closer proximity to the base of domain I.

Protein synthesis is principally regulated at the initiation stage (rather than during elongation or termination), allowing rapid, reversible and spatial control over gene expression. Progress over recent years in determining the structures and activities of initiation factors, and in mapping their interactions within ribosomal initiation complexes, has significantly advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studies of regulation of initiation by mechanisms that include modulation of the activity of initiation factors (which affects almost all scanning-dependent initiation), or via sequence-specific RNA-binding proteins and microRNAs (which thus impact individual mRNAs).

Translational control plays an important role in cell growth and tumorigenesis. Cap-dependent translation initiation of mammalian mRNAs with structured 5′UTRs requires the DExH-box protein, DHX29, in vitro. Here we show that DHX29 is important for translation in vivo. Down-regulation of DHX29 leads to impaired translation, resulting in disassembly of polysomes and accumulation of mRNA-free 80S monomers. DHX29 depletion also impedes cancer cell growth in culture and in xenografts. Thus, DHX29 is a bona fide translation initiation factor that potentially can be exploited as a target to inhibit cancer cell growth.

Viral internal ribosomal entry sites (IRESs) mediate end-independent translation initiation. There are 4 major structurally-distinct IRES groups: type 1 (e.g., poliovirus) and type 2 (e.g., encephalomyocarditis virus), which are dissimilar except for a Yn-Xm-AUG motif at their 3′ borders, type 3 (e.g., hepatitis C virus), and type 4 (dicistroviruses). Type 2–4 IRESs mediate initiation by distinct mechanisms that are nevertheless all based on specific noncanonical interactions with canonical components of the translation apparatus, such as eukaryotic initiation factor (eIF) 4G (type 2), 40S ribosomal subunits (types 3 and 4), and eIF3 (type 3). The mechanism of initiation on type 1 IRESs is unknown. We now report that domain V of type 1 IRESs, which is adjacent to the Yn-Xm-AUG motif, specifically interacts with the central domain of eIF4G. The position and orientation of eIF4G relative to the Yn-Xm-AUG motif is analogous in type 1 and 2 IRESs. eIF4G promotes recruitment of eIF4A to type 1 IRESs, and together, eIF4G and eIF4A induce conformational changes at their 3′ borders. The ability of mutant type 1 IRESs to bind eIF4G/eIF4A correlated with their translational activity. These characteristics parallel the mechanism of initiation on type 2 IRESs, in which the key event is binding of eIF4G to the J–K domain adjacent to the Yn-Xm-AUG motif, which is enhanced by eIF4A. These data suggest that fundamental aspects of the mechanisms of initiation on these unrelated classes of IRESs are similar.

Specific interactions of the classical swine fever virus internal ribosomal entry site (IRES) with 40S ribosomal subunits and eukaryotic translation initiation factor (eIF)3 enable 43S preinitiation complexes containing eIF3 and eIF2–GTP–Met‐tRNA Met i to bind directly to the initiation codon, yielding 48S initiation complexes. We report that eIF5B or eIF5B/eIF3 also promote Met‐tRNA Met i binding to IRES–40S complexes, forming 48S complexes that can assemble elongation‐competent ribosomes. Although 48S complexes assembled both by eIF2/eIF3‐ and eIF5B/eIF3‐mediated Met‐tRNA Met i recruitment were destabilized by eIF1, dissociation of 48S complexes formed with eIF2 could be out‐competed by efficient subunit joining. Deletion of IRES domain II, which is responsible for conformational changes induced in 40S subunits by IRES binding, eliminated the sensitivity of 48S complexes assembled by eIF2/eIF3‐ and eIF5B/eIF3‐mediated mechanisms to eIF1‐induced destabilization. However, 48S complexes formed by the eIF5B/eIF3‐mediated mechanism on the truncated IRES could not undergo efficient subunit joining, as reported previously for analogous complexes assembled with eIF2, indicating that domain II is essential for general conformational changes in 48S complexes, irrespective of how they were assembled, that are required for eIF5‐induced hydrolysis of eIF2‐bound GTP and/or subunit joining.

The meeting on Translational Control and Non‐Coding RNA took place between 8 and 12 November 2006, in Nove Hrady, the Czech Republic, and was organized by M. Pospisek and L. Valasek. ![][1] Many pathways that regulate gene expression target the complex process of protein synthesis and in particular its initiation stage. During the past 40 years or so, all the initiation factors required for the assembly of translationally competent ribosomes have been identified and their principal roles determined. Recently, there has been significant progress in determining the structure of the components of the translation apparatus: from crystal structures of prokaryotic ribosomes, to crystal and nuclear magnetic resonance structures of factors, and cryo‐electron microscopy reconstructions of functional ribosomal complexes. These advances now allow the molecular mechanisms of the stages involved in initiation to be investigated in detail. Concurrently, diverse studies have revealed that translational control regulates processes ranging from development to learning. This meeting highlighted recent advances about canonical and internal ribosome entry site (IRES)‐mediated mechanisms of eukaryotic initiation, and the control of translation during development in response to stress and by mechanisms ranging from signal transduction pathways to regulation by noncoding RNAs and RNA‐binding proteins. Canonical 5′‐end‐dependent eukaryotic translation initiation requires at least 11 initiation factors (eIFs) and occurs in two stages: formation of the 48S initiation complex at the AUG codon of messenger RNA (mRNA), and its joining with a 60S ribosomal subunit (Fig 1; Hinnebusch et al , 2007; Pestova et al , 2007). eIF1 has a crucial role in the selection of initiation codons, allowing scanning 43S complexes to discriminate against codon–anticodon mismatches and preventing premature eIF5‐induced hydrolysis of eIF2‐bound GTP and Pi release. According to a recent model, eIF1—in cooperation with eIF1A—promotes a scanning‐competent ‘open’ conformation of the 43S complex. The establishment of codon–anticodon base‐pairing leads … [1]: /embed/graphic-1.gif

Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that mediates displacement of initiation factors from the 40S ribosomal subunit in 48S initiation complexes and joining of 40S and 60S subunits. Here, we determined eIF5B's position on 80S ribosomes by directed hydroxyl radical cleavage. In the resulting model, eIF5B is located in the intersubunit cleft of the 80S ribosome: domain 1 is positioned near the GTPase activating center of the 60S subunit, domain 2 interacts with the 40S subunit (helices 3, 5 and the base of helix 15 of 18S rRNA and ribosomal protein (rp) rpS23), domain 3 is sandwiched between subunits and directly contacts several ribosomal elements including Helix 95 of 28S rRNA and helix 44 of 18S rRNA, domain 4 is near the peptidyl-transferase center and its helical subdomain contacts rpL10E. The cleavage data also indicate that binding of eIF5B might induce conformational changes in both subunits, with ribosomal segments wrapping around the factor. Some of these changes could also occur upon binding of other translational GTPases, and may contribute to factor recognition.

Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that is responsible for the final step in initiation, which involves the displacement of initiation factors from the 40S ribosomal subunit in initiation complexes and its joining with the 60S subunit. Hydrolysis of eIF5B-bound GTP is not required for its function in subunit joining but is necessary for the subsequent release of eIF5B from assembled 80S ribosomes. Here we investigated the kinetics of guanine nucleotide binding to eIF5B by a fluorescent stopped-flow technique using fluorescent mant derivatives of GTP and GDP and of the GTP analogues GTPgammaS and GMPPNP. The affinity of eIF5B for mant-GTP (Kd approximately 14-18 microM) was approximately 7-fold less than for mant-GDP (Kd approximately 2.3 microM), and both guanine nucleotides dissociated rapidly from eIF5B (k-1mant-GTP approximately 22-28 s-1, k-1mant-GDP approximately 10-14 s-1). These properties of eIF5B suggest a rapid spontaneous GTP/GDP exchange on eIF5B and are therefore consistent with it having no requirement for a special guanine nucleotide exchange factor. The affinity of eIF5B for mant-GTPgammaS was about 2 times lower (Kd approximately 6.9 microM) and for mant-GMPPNP 1.5 times higher (Kd approximately 25.7 microM) than for mant-GTP, indicating that eIF5B tolerates modifications of the triphosphate moiety well.

Protein translation typically begins with the recruitment of the 43S ribosomal complex to the 5' cap of mRNAs by a cap-binding complex. However, some transcripts are translated in a cap-independent manner through poorly understood mechanisms. Here, we show that mRNAs containing N(6)-methyladenosine (m(6)A) in their 5' UTR can be translated in a cap-independent manner. A single 5' UTR m(6)A directly binds eukaryotic initiation factor 3 (eIF3), which is sufficient to recruit the 43S complex to initiate translation in the absence of the cap-binding factor eIF4E. Inhibition of adenosine methylation selectively reduces translation of mRNAs containing 5'UTR m(6)A. Additionally, increased m(6)A levels in the Hsp70 mRNA regulate its cap-independent translation following heat shock. Notably, we find that diverse cellular stresses induce a transcriptome-wide redistribution of m(6)A, resulting in increased numbers of mRNAs with 5' UTR m(6)A. These data show that 5' UTR m(6)A bypasses 5' cap-binding proteins to promote translation under stresses.

Translation initiation in eukaryotes leads to the assembly of an 80S ribosome containing aminoacylated initiator tRNA (Met-) in the P (peptidyl) site with its anticodon base-paired to the initiation codon of mRNA. It involves at least eleven eukaryotic initiation factors (eIFs). eIF2, GTP, and Met- form a ternary complex, which together with eIF3, eIF1, and eIF1A binds to the 40S subunit to form a 43S complex (Benne and Hershey 1978). After binding to the 5′-end of mRNA, mediated by eIF4A, eIF4B, and eIF4F, the 43S complex scans downstream until it encounters an AUG triplet in a favorable context, where it stops and forms a stable 48S complex with established codon-anticodon base-pairing. eIF1 plays a key role in initiation codon selection (Yoon and Donahue 1992; Pestova et al. 1998; Pestova and Kolupaeva 2002). Finally, a 60S subunit joins the 48S complex but cannot do so directly, presumably because the interface surface of the 40S subunit is occupied by eIF1, eIF1A, eIF2, and eIF3, which must be displaced. The first step in ribosomal subunit joining is hydrolysis of eIF2-bound GTP and release of eIF2-GDP from 48S complexes (Das and Maitra 2002). eIF2 consists of α, β, and γ subunits. The structure of eIF2γ is characteristic of GTP-binding proteins (Schmitt et al. 2002), but eIF2 does not have intrinsic GTPase activity. Hydrolysis of eIF2-bound GTP is induced by eIF5, a GTPase-activating protein (GAP) specific for eIF2, which provides an “arginine finger” for the catalytic center of eIF2γ (Das and Maitra 2002). eIF5 binds directly to eIF2β but induces the GTPase activity of eIF2 only in those eIF2 ternary complexes that are bound to 40S subunits (Raychaudhuri et al. 1985). A few biochemical experiments indicate that eIF5-induced hydrolysis of eIF2-bound GTP may be weakly AUG-dependent (e.g., Raychaudhuri et al. 1985) but they were done in a minimal system containing only 40S subunits, eIF2 ternary complex, and eIF5, in which AUG triplets also stimulated the prior stage, binding of ternary complexes to 40S subunits. Although in vitro data concerning a link between initiation codon recognition and hydrolysis of eIF2-bound GTP are limited, strong genetic data support the importance of a stringent connection between them: Mutations in eIF5 (which increase its GAP activity), in eIF2γ (which increase eIF2 dissociation from Met- even without GTP hydrolysis) and in eIF2β (which increase eIF2's intrinsic GTPase activity) all enhance initiation at non-AUG codons in yeast (Huang et al. 1997). Thus premature hydrolysis of eIF2-bound GTP and release of eIF2 from initiation complexes reduce the fidelity of initiation and should be avoided. It has been widely assumed that eIF5-induced hydrolysis of eIF2-bound GTP and release of eIF2-GDP from 48S complexes lead to release of all other factors (e.g., Das and Maitra 2002). If this were so, hydrolysis of eIF2-bound GTP should logically suffice for subunit joining. However, it is sufficient only for 60S subunits to join minimal 48S complexes formed on AUG triplets, but not to 48S complexes formed on native mRNA with eIF2, eIF3, eIF1, and eIF1A. In this case, eIF5B is also required (Pestova et al. 2000). It has been suggested that eIF5B's role could be to adjust the position of Met- on the 40S subunit as a prerequisite for subunit joining (Roll-Mecak et al. 2001). However, eIF5B's role in subunit joining could also be because, contrary to the common assumption, one or more of eIF1, eIF1A, and eIF3 are not displaced from the 40S subunit after eIF5-induced eIF2-GDP release. Although eIF1, eIF1A, and eIF3 can all bind to 40S subunits independent of the eIF2 ternary complex (Fraser et al. 2004; Maag and Lorsch 2003), contrary to other reports (e.g., Trachsel and Staehelin 1979), in the absence of eIF2 ternary complex, these factors do not protect 40S subunits from joining with 60S subunits (Chaudhuri et al. 1999; Kolupaeva et al. 2005). Therefore, even if eIF1, eIF1A, and eIF3 were not released from 48S complexes after GTP hydrolysis, their presence should not prevent subunit joining. However, we recently found that eIF3 stably binds to 40S subunits in the presence of single-stranded RNA, forming ternary complexes in which RNA is most likely bound in the mRNA-binding cleft of the 40S subunit, and this prevents 40S subunits from joining with 60S subunits even in the absence of eIF2 ternary complex (Kolupaeva et al. 2005). These data suggest that association of eIF3 with 40S subunits in mRNA-containing 48S complexes after release of eIF2-GDP would potentially block subunit joining, and the role of eIF5B could be to displace eIF3 and possibly eIF1 and eIF1A from the 40S subunit after release of eIF2. This could occur in at least two ways. eIF5B could either actively displace these factors, yielding a free 40S subunit/Met- complex before subunit joining, or it could mediate subunit joining on 48S complexes containing eIF3, eIF1, and eIF1A so that these factors dissociate during the actual subunit joining event. We have investigated these questions and report here that eIF5-induced hydrolysis of eIF2-bound GTP in 48S complexes assembled on mRNA led to release of only eIF2, whereas eIF1 and eIF3 remained bound to 40S subunits. eIF5B does not influence factor displacement in the absence of 60S subunits, and eIF3 and eIF1 are released only during the actual subunit joining event mediated by eIF5B. In the absence of eIF1, eIF5-stimulated hydrolysis of eIF2-bound GTP did not depend on establishment of codon-anticodon interaction and occurred at the same rate in 43S and 48S complexes. In the presence of eIF1, GTP hydrolysis in 43S complexes was much slower than in 43S or 48S complexes assembled in the absence of eIF1. Establishment of codon-anticodon interaction in 48S complexes assembled in the presence of eIF1 relieved eIF1's inhibition. eIF1 therefore plays the role of a negative regulator, which inhibits premature GTP hydrolysis and links codon-anticodon base-pairing with hydrolysis of eIF2-bound GTP. Thus, in addition to its role in initiation codon selection at the stage of 48S complex formation, eIF1 also participates in ensuring the fidelity of initiation at the stage of hydrolysis of eIF2-bound GTP.

The cricket paralysis virus intergenic region internal ribosomal entry site (CrPV IGR IRES) can assemble translation initiation complexes by binding to 40S subunits without Met-tRNA(Met)(i) and initiation factors (eIFs) and then by joining directly with 60S subunits, yielding elongation-competent 80S ribosomes. Here, we report that eIF1, eIF1A and eIF3 do not significantly influence IRES/40S subunit binding but strongly inhibit subunit joining and the first elongation cycle. The IRES can avoid their inhibitory effect by its ability to bind directly to 80S ribosomes. The IRES's ability to bind to 40S subunits simultaneously with eIF1 allowed us to use directed hydroxyl radical cleavage to map its position relative to the known position of eIF1. A connecting loop in the IRES's pseudoknot (PK) III domain, part of PK II and the entire domain containing PK I are solvent-exposed and occupy the E site and regions of the P site that are usually occupied by Met-tRNA(Met)(i).

The translation initiation factors (IFs) IF1/eIF1A and IF2/eIF5B have been conserved throughout all kingdoms. Although the central roles of the bacterial factors IF1 and IF2 were established long ago, the importance of their eukaryotic homologs, eukaryotic IFs (eIFs) eIF1A and eIF5B, has only recently become evident. The translation machinery in eukaryotes is more complex and accordingly, eIF1A and eIF5B seem to have acquired a number of new functions while also retaining many of the roles of bacterial IF1 and IF2. IF1 and IF2 have been shown to interact on the ribosome but no binding has been detected for the free factors. In contrast, yeast eIF1A and eIF5B have been reported to interact in the absence of ribosomes. Here, we have identified the binding interface between human eIF1A and the C-terminal domain of eIF5B by using solution NMR. That interaction interface involves the C termini of the two proteins, which are not present in bacterial IF1 and IF2. The interaction is, therefore, unique to eukaryotes. A structural model for the interaction of eIF1A and eIF5B in the context of the ribosome is presented. We propose that eIF1A and eIF5B simultaneously interact at two sites that are >50 Å apart: through their C termini as reported here, and through an interface previously identified in bacterial IF1 and IF2. The binding between the C termini of eIF1A and eIF5B has implications for eukaryote-specific mechanisms of recruitment and release of translation IFs from the ribosome.

Reconstitution of translation elongation from purified components confirmed that ribosomes that assembled on the Cricket paralysis virus intercistronic internal ribosomal entry site (IRES) without the involvement of initiation factors or initiator tRNA were active in elongation and are, therefore, true initiation complexes. The first elongation cycle occurred without peptide bond formation on 80S ribosomes that did not contain tRNA in the P site. It required elongation factors 1A and 2 and A site-cognate aminoacylated tRNA. Cycloheximide arrested ribosomes on the IRES only after two cycles of elongation, when the first deacylated tRNA reached the E-site after translocation from the A-site.

Initiation factors IF2 in bacteria and eIF2 in eukaryotes are GTPases that bind Met-tRNA \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{Met}\end{equation*}\end{document} to the small ribosomal subunit. eIF5B, the eukaryotic ortholog of IF2, is a GTPase that promotes ribosomal subunit joining. Here we show that eIF5B GTPase activity is required for protein synthesis. Mutation of the conserved Asp-759 in human eIF5B GTP-binding domain to Asn converts eIF5B to an XTPase and introduces an XTP requirement for subunit joining and translation initiation. Thus, in contrast to bacteria where the single GTPase IF2 is sufficient to catalyze translation initiation, eukaryotic cells require hydrolysis of GTP by both eIF2 and eIF5B to complete translation initiation.

To elucidate an outline of the mechanism of eukaryotic translation initiation, 48S complex formation was analyzed on defined mRNAs in reactions reconstituted in vitro from fully purified translation components. We found that a ribosomal 40S subunit, eukaryotic initiation factor (eIF) 3, and the eIF2 ternary complex form a 43S complex that can bind to the 5′-end of an unstructured 5′-untranslated region (5′-UTR) and in the presence of eIF1 scan along it and locate the initiation codon without a requirement for adenosine triphosphate (ATP) or factors (eIF4A, eIF4B, eIF4F) associated with ATP hydrolysis. Scanning on unstructured 5′-UTRs was enhanced by ATP, eIFs 4A and 4B, and the central domain of the eIF4G subunit of eIF4F. Their omission increased the dependence of scanning on eIFs 1 and 1A. Ribosomal movement on 5′-UTRs containing even weak secondary structures required ATP and RNA helicases. eIF4F was essential for scanning, and eIFs 4A and 4B were insufficient to promote this process in the absence of eIF4F. We report that in addition to its function in scanning, eIF1 also plays a principal role in initiation codon selection. In the absence of eIF1, 43S complexes could no longer discriminate between cognate and noncognate initiation codons or sense the nucleotide context of initiation codons and were able to assemble 48S complexes on 5′-proximal AUG triplets located only 1, 2, and 4 nt from the 5′-end of mRNA.

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5′ end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.

A complex of eukaryotic initiation factors (eIFs) 4A, 4E, and 4G (collectively termed eIF4F) plays a key role in recruiting mRNAs to ribosomes during translation initiation. The site of ribosomal entry onto most mRNAs is determined by interaction of the 5'-terminal cap with eIF4E; eIFs 4A and 4G may facilitate ribosomal entry by modifying mRNA structure near the cap and by interacting with ribosome-associated factors. eIF4G recruits uncapped encephalomyocarditis virus (EMCV) mRNA to ribosomes without the involvement of eIF4E by binding directly to the approximately 450-nucleotide long EMCV internal ribosome entry site (IRES). We have used chemical and enzymatic probing to map the eIF4G binding site to a structural element within the J-K domain of the EMCV IRES that consists of an oligo(A) loop at the junction of three helices. The oligo(A) loop itself is not sufficient to form stable complexes with eIF4G since alteration of its structural context abolished its interaction with eIF4G. Addition of wild type or trans-dominant mutant forms of eIF4A to binary IRES.eIF4G complexes did not further alter the pattern of chemical/enzymatic modification of the IRES.

Translation of hepatitis C virus (HCV) and classical swine fever virus (CSFV) RNAs is initiated by cap-independent attachment (internal entry) of ribosomes to the ∼350-nucleotide internal ribosomal entry segment (IRES) at the 5′ end of both RNAs. Eukaryotic initiation factor 3 (eIF3) binds specifically to HCV and CSFV IRESs and plays an essential role in the initiation process on them. Here we report the results of chemical and enzymatic footprinting analyses of binary eIF3-IRES complexes, which have been used to identify the eIF3 binding sites on HCV and CSFV IRESs. eIF3 protected an internal bulge in the apical stem IIIb of domain III of the CSFV IRES from chemical modification and protected bonds in and adjacent to this bulge from cleavage by RNases ONE and V 1 . eIF3 protected an analagous region in domain III of the HCV IRES from cleavage by these enzymes. These results are consistent with the results of primer extension analyses and were supported by observations that deletion of stem-loop IIIb or of the adjacent hairpin IIIc from the HCV IRES abrogated the binding of eIF3 to this RNA. This is the first report that eIF3 is able to bind a eukaryotic mRNA in a sequence- or structure-specific manner. UV cross-linking of eIF3 to [ 32 P]UTP-labelled HCV and CSFV IRES elements resulted in strong labelling of 4 (p170, p116, p66, and p47) of the 10 subunits of eIF3, 1 or more of which are likely to be determinants of these interactions. In the cytoplasm, eIF3 is stoichiometrically associated with free 40S ribosomal subunits. The results presented here are consistent with a model in which binding of these two translation components to separate, specific sites on both HCV and CSFV IRESs enhances the efficiency and accuracy of binding of these RNAs to 40S subunits in an orientation that promotes entry of the initiation codon into the ribosomal P site.

Initiation of translation of hepatitis C virus and classical swine fever virus mRNAs results from internal ribosomal entry. We reconstituted internal ribosomal entry in vitro from purified translation components and monitored assembly of 48S ribosomal preinitiation complexes by toe-printing. Ribosomal subunits (40S) formed stable binary complexes on both mRNAs. The complex structure of these RNAs determined the correct positioning of the initiation codon in the ribosomal “P” site in binary complexes. Ribosomal binding and positioning on these mRNAs did not require the initiation factors eIF3, eIF4A, eIF4B, and eIF4F and translation of these mRNAs was not inhibited by a trans-dominant eIF4A mutant. Addition of Met–tRNAiMet, eIF2, and GTP to these binary ribosomal complexes resulted in formation of 48S preinitiation complexes. The striking similarities between this eukaryotic initiation mechanism and the mechanism of translation initiation in prokaryotes are discussed.

Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5'-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5-7, 9-12), but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF3 and the HCV IRES revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S-IRES binary complex, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favouring translation of viral mRNAs.

Summary Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, methionylated initiator methionine transfer RNA (Met-tRNA i Met ), eukaryotic initiation factor (eIF) 2, and guanosine triphosphate form a ternary complex (TC). The TC, eIF3, eIF1, and eIF1A cooperatively bind to the 40S subunit, yielding the 43S preinitiation complex, which is ready to attach to messenger RNA (mRNA) and start scanning to the initiation codon. Scanning on structured mRNAs additionally requires DHX29, a DExH-box protein that also binds directly to the 40S subunit. Here, we present a cryo-electron microscopy structure of the mammalian DHX29-bound 43S complex at 11.6 A resolution. It reveals that eIF2 interacts with the 40S subunit via its α subunit and supports Met-tRNA i Met in an unexpected P/I orientation (eP/I). The structural core of eIF3 resides on the back of the 40S subunit, establishing two principal points of contact, whereas DHX29 binds around helix 16. The structure provides insights into eukaryote-specific aspects of translation, including the mechanism of action of DHX29.

Translation of picornavirus RNA is initiated after ribosomal binding to an internal ribosomal entry site (IRES) within the 5' untranslated region. We have reconstituted IRES-mediated initiation on encephalomyocarditis virus RNA from purified components and used primer extension analysis to confirm the fidelity of 48S preinitiation complex formation. Eukaryotic initiation factor 2 (eIF2), eIF3, and eIF4F were required for initiation; eIF4B and to a lesser extent the pyrimidine tract-binding protein stimulated this process. We show that eIF4F binds to the IRES in a novel cap-independent manner and suggest that cap- and IRES-dependent initiation mechanisms utilize different modes of interaction with this factor to promote ribosomal attachment to mRNA.

Eukaryotic translation is initiated following binding of ribosomes either to the capped 5' end of an mRNA or to an internal ribosomal entry site (IRES) within its 5' nontranslated region. These processes are both mediated by eukaryotic initiation factor 4F (eIF4F), which consists of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G subunits. Here we present a functional analysis of eIF4F which defines the subunits and subunit domains necessary for its function in initiation mediated by the prototypical IRES element of encephalomyocarditis virus. In an initiation reaction reconstituted in vitro from purified translation components and lacking eIF4A and -4F, IRES-mediated initiation did not require the cap-binding protein eIF4E but was absolutely dependent on eIF4A and the central third of eIF4G. This central domain of eIF4G bound strongly and specifically to a structural element within the encephalomyocarditis virus IRES upstream of the initiation codon in an ATP-independent manner and with the same specificity as eIF4F. The carboxy-terminal third of eIF4G did not bind to the IRES. The central domain of eIF4G was itself UV cross-linked to the IRES and strongly stimulated UV cross-linking of eIF4A to the IRES in conjunction with either eIF4B or with the carboxy-terminal third of eIF4G.

The cadicivirus IRES diverges structurally from canonical Type 1 IRESs (e.g. poliovirus) but nevertheless also contains an essential GNRA tetraloop in a subdomain (d10c) that is homologous to poliovirus dIVc. In addition to canonical initiation factors, the canonical Type 1 and divergent cadicivirus IRESs require the same IRES trans-acting factor, poly(C)-binding protein 2 (PCBP2). PCBP2 has three KH domains and binds poliovirus IRES domain dIV in the vicinity of the tetraloop. How PCBP2 binds the cadicivirus IRES, and the roles of PCBP2 and the tetraloop in Type 1 IRES function are unknown. Here, directed hydroxyl radical probing showed that KH1 also binds near the cadicivirus tetraloop. KH2 and KH3 bind adjacently to an IRES subdomain (d10b) that is unrelated to dIV, with KH3 in an inverted orientation. KH3 is critical for PCBP2's binding to this IRES whereas KH1 is essential for PCBP2's function in promoting initiation. PCBP2 enforced the wild-type structure of d10c when it contained minor destabilizing substitutions, exposing the tetraloop. Strikingly, PCBP2 enhanced initiation on mutant IRESs that retained consensus GNRA tetraloops, whereas mutants with divergent sequences did not respond to PCBP2. These studies show that PCBP2 enables the IRES to exploit the GNRA tetraloop to enhance initiation.

Translation termination in eukaryotes occurs in response to a stop codon in the ribosomal A-site and requires two release factors (RFs), eRF1 and eRF3, which bind to the A-site as an eRF1/eRF3/GTP complex with eRF1 responsible for codon recognition. After GTP hydrolysis by eRF3, eRF1 triggers hydrolysis of the polypeptidyl-tRNA, releasing the completed protein product. This leaves an 80S ribosome still bound to the mRNA, with deacylated tRNA in its P-site and at least eRF1 in its A-site, which needs to be disassembled and released from the mRNA to allow further rounds of translation. The first step in recycling is dissociation of the 60S ribosomal subunit, leaving a 40S/deacylated tRNA complex bound to the mRNA. This is mediated by ABCE1, which is a somewhat unusual member of the ATP-binding cassette family of proteins with no membrane-spanning domain but two essential iron-sulfur clusters. Two distinct pathways have been identified for subsequent ejection of the deacylated tRNA followed by dissociation of the 40S subunit from the mRNA, one executed by a subset of the canonical initiation factors (which therefore starts the process of preparing the 40S subunit for the next round of translation) and the other by Ligatin or homologous proteins. However, although this is the normal sequence of events, there are exceptions where the termination reaction is followed by reinitiation on the same mRNA (usually) at a site downstream of the stop codon. The overwhelming majority of such reinitiation events occur when the 5'-proximal open reading frame (ORF) is short and can result in significant regulation of translation of the protein-coding ORF, but there are also rare examples, mainly bicistronic viral RNAs, of reinitiation after a long ORF. Here, we review our current understanding of the mechanisms of termination, ribosome recycling, and reinitiation after translation of short and long ORFs.

Protein synthesis is a cyclical process, consisting of initiation, elongation, termination and ribosome recycling stages. Initiation requires pools of separated small and large ribosomal subunits, and it has been known for about forty years that, after termination, eukaryotic 80S ribosomes dissociate from polysomes into 40S and 60S subunits, which then either participate in the next round of initiation, or enter a reservoir of stable, translationally inactive 80S monosomes (Adamson et al., 1969; Hogan and Korner, 1968b; Kaempfer 1969; Falvey and Staehelin, 1970; Howard et al., 1970; Henshaw et al., 1973). Although 80S monosomes accumulate in response to stresses that inhibit initiation, such as amino acid or glucose starvation, reversal of these conditions allows them to be dissociated and their subunits to re-enter the translation process (Hogan and Korner, 1968 a). In bacteria, recycling of post-termination 70S ribosomal complexes (post-TCs) requires elongation factor EF-G, the dedicated ribosome recycling factor RRF (Hirashima and Kaji, 1973) and initiation factor IF3.

SummaryEukaryotic protein synthesis begins with assembly of 48S initiation complexes at the initiation codon of mRNA, which requires at least seven initiation factors (eIFs). First, 43S preinitiation complexes comprising 40S ribosomal subunits, eIFs 3, 2, 1, and 1A, and tRNAMeti attach to the 5′-proximal region of mRNA and then scan along the 5′ untranslated region (5′UTR) to the initiation codon. Attachment of 43S complexes is mediated by three other eIFs, 4F, 4A, and 4B, which cooperatively unwind the cap-proximal region of mRNA and later also assist 43S complexes during scanning. We now report that these seven eIFs are not sufficient for efficient 48S complex formation on mRNAs with highly structured 5′UTRs, and that this process requires the DExH-box protein DHX29. DHX29 binds 40S subunits and hydrolyzes ATP, GTP, UTP, and CTP. NTP hydrolysis by DHX29 is strongly stimulated by 43S complexes and is required for DHX29's activity in promoting 48S complex formation.

Selection of the AUG start codon is a key step in translation initiation requiring hydrolysis of GTP in the eIF2•GTP•Met-tRNAiMet ternary complex (TC) and subsequent Pi release from eIF2•GDP•Pi. It is thought that eIF1 prevents recognition of non-AUGs by promoting scanning and blocking Pi release at non-AUG codons. We show that Sui− mutations in Saccharomyces cerevisiae eIF1, which increase initiation at UUG codons, reduce interaction of eIF1 with 40S subunits in vitro and in vivo, and both defects are diminished in cells by overexpressing the mutant proteins. Remarkably, Sui− mutation ISQLG93–97ASQAA (abbreviated 93–97) accelerates eIF1 dissociation and Pi release from reconstituted preinitiation complexes (PICs), whereas a hyperaccuracy mutation in eIF1A (that suppresses Sui− mutations) decreases the eIF1 off-rate. These findings demonstrate that eIF1 dissociation is a critical step in start codon selection, which is modulated by eIF1A. We also describe Gcd− mutations in eIF1 that impair TC loading on 40S subunits or destabilize the multifactor complex containing eIF1, eIF3, eIF5, and TC, showing that eIF1 promotes PIC assembly in vivo beyond its important functions in AUG selection.

Bacterial translation initiation factor IF1 is an S1 domain protein that belongs to the oligomer binding (OB) fold proteins. Cold shock domain (CSD)-containing proteins such as CspA (the major cold shock protein of Escherichia coli ) and its homologues also belong to the OB fold protein family. The striking structural similarity between IF1 and CspA homologues suggests a functional overlap between these proteins. Certain members of the CspA family of cold shock proteins act as nucleic acid chaperones: they melt secondary structures in nucleic acids and act as transcription antiterminators. This activity may help the cell to acclimatize to low temperatures, since cold-induced stabilization of secondary structures in nascent RNA can impede transcription elongation. Here we show that the E. coli translation initiation factor, IF1, also has RNA chaperone activity and acts as a transcription antiterminator in vivo and in vitro. We further show that the RNA chaperone activity of IF1, although critical for transcription antitermination, is not essential for its role in supporting cell growth, which presumably functions in translation. The results thus indicate that IF1 may participate in transcription regulation and that cross talk and/or functional overlap may exist between the Csp family proteins, known to be involved in transcription regulation at cold shock, and S1 domain proteins, known to function in translation.

The canonical initiation process is the most complex aspect of translation in eukaryotes. It involves the coordinated interactions of at least 11 eukaryotic initiation factors, 40S and 60S ribosomal subunits, mRNA, and aminoacylated initiator tRNA (Met-tRNA(i)(Met)), as well as binding and hydrolysis of GTP and ATP. The factor requirements for many individual steps in this process, including scanning, initiation codon recognition, and ribosomal subunit joining, have until recently been obscure. We established the factor requirements for these steps by reconstituting the initiation process in vitro from individual purified components of the translation apparatus and developed approaches to explain the mechanism of individual steps and the roles of individual factors and to characterize the structure of initiation complexes. Here we describe protocols for the purification of native initiation factors and for expression and purification of active recombinant forms of all single subunit initiation factors, for the reconstitution of the initiation process, and for determination of the position of ribosomal complexes on mRNA by primer extension inhibition ("toe printing"). We also describe protocols for site-directed ultraviolet (UV) cross-linking to determine the interactions of individual nucleotides in mRNA with components of the initiation complex and for directed hydroxyl radical probing to determine the position of initiation factors on the ribosome.

The internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV) and related viruses promote initiation of translation by a noncanonical end-independent mechanism. To characterize this mechanism at the molecular level, we have developed biochemical approaches to reconstitute the process in vitro from individual purified components of the translation apparatus, developed methods to characterize steps in this process so that the functions of individual proteins can be characterized, and adapted assays such as primer extension inhibition ("toe printing") to monitor accurate assembly on the IRES of ribosomal 48S and 80S complexes. In vitro reconstitution of 48S complex formation offers an approach for the functional identification of IRES trans-acting factors (ITAFs) that are required for initiation in addition to canonical initiation factors and revealed that despite being related, different EMCV-like IRESs nevertheless have distinct ITAF requirements. Toe printing revealed that a common feature of initiation on EMCV-like IRESs is the stable binding of an eIF4G/eIF4A complex to them near the initiation codon, where it can locally unwind RNA to facilitate ribosomal attachment. The same toe printing assay indicated that binding of ITAFs to these IRESs enhances binding of these two canonical initiation factors. We also describe protocols for chemical and enzymatic footprinting to determine the interactions of trans-acting factors with the IRES at nucleotide resolution and for directed hydroxyl radical probing to determine their orientation on the IRES.

Initiation is the first phase in protein synthesis, during which eukaryotic initiation factors assemble an 80S ribosome at an initiation codon of a messenger RNA (mRNA) such that this triplet is base-paired to the CAU anticodon of aminoacylated initiator methionyl-transfer RNA in the ribosomal peptidyl site. Initiation is followed by the elongation phase, in which the 80S ribosome moves along the mRNA, translating the open reading frame into protein. Keywords: eukaryotic translation initiation factor; messenger ribonucleic acid; ribosome; transfer ribonucleic acid; translation initiation

International audience; 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.

Eukaryotic initiation factor (eIF) eIF1 maintains the fidelity of initiation codon selection by enabling 43S complexes to reject codon–anticodon mismatches, to recognize the initiation codon context, and to discriminate against establishing a codon–anticodon interaction with AUGs located

Initiation of translation of encephalomyocarditis virus mRNA is mediated by an internal ribosome entry site (IRES) comprising structural domains H, I, J-K, and L immediately upstream of the initiation codon AUG at nucleotide 834 (AUG834). Assembly of 48S ribosomal complexes on the IRES requires eukaryotic initiation factor 2 (eIF2), eIF3, eIF4A, and the central domain of eIF4G to which eIF4A binds. Footprinting experiments confirmed that eIF4G binds a three-way helical junction in the J-K domain and showed that it interacts extensively with RNA duplexes in the J-K and L domains. Deletion of apical hairpins in the J and K domains synergistically impaired the binding of eIF4G and IRES function. Directed hydroxyl radical probing, done by using Fe(II) tethered to surface residues in eIF4G's central domain, indicated that it is oriented with its N terminus towards the base of domain J and its C terminus towards the apex. eIF4G recruits eIF4A to a defined location on the IRES, and the eIF4G/eIF4A complex caused localized ATP-independent conformational changes in the eIF4G-binding region of the IRES. This complex also induced more extensive conformational rearrangements at the 3' border of the ribosome binding site that required ATP and active eIF4A. We propose that these conformational changes prepare the region flanking AUG834 for productive binding of the ribosome.

Most eukaryotic mRNAs require the cap-binding complex elF4F for efficient initiation of translation, which occurs as a result of ribosomal scanning from the capped 5' end of the mRNA to the initiation codon. A few cellular and viral mRNAs are translated by a cap and end-independent mechanism known as internal ribosomal entry. The internal ribosome entry site (IRES) of classical swine fever virus (CSFV) is approximately 330 nt long, highly structured, and mediates internal initiation of translation with no requirement for elF4F by recruiting a ribosomal 43S preinitiation complex directly to the initiation codon. The key interaction in this process is the direct binding of ribosomal 40S subunits to the IRES to form a stable binary complex in which the initiation codon is positioned precisely in the ribosomal P site. Here, we report the results of analyses done using enzymatic footprinting and mutagenesis of the IRES to identify structural components in it responsible for precise binding of the ribosome. Residues flanking the initiation codon and extending from nt 363-391, a distance equivalent to the length of the 40S subunit mRNA-binding cleft, were strongly protected from RNase cleavage, as were nucleotides in the adjacent pseudoknot and in the more distal subdomain IIId1. Ribosomal binding and IRES-mediated initiation were abrogated by disruption of helix 1b of the pseudoknot and very severely reduced by mutation of the protected residues in IIId1 and by disruption of domain IIIa. These observations are consistent with a model for IRES function in which binding of the region flanking the initiation codon to the decoding region of the ribosome is determined by multiple additional interactions between the 40S subunit and the IRES.

The initiation phase of translation in eukaryotes is the process leading to assembly of a translation-competent 80S ribosome at the initiation codon of an mRNA. The canonical initiation process involves more than 10 initiation factors (6, 27, 38). The first stage in the initiation process is the binding of a eukaryotic initiation factor 2 (eIF2)-GTP-initiator tRNA complex, eIF1A, and eIF3 to the 40S ribosomal subunit to form a 43S complex. The second stage involves the binding of mRNA to this ribosomal complex and involves eIF3, eIF4A, eIF4B, eIF4F, and the poly(A)-binding protein (PABP). All nonorganellar cellular mRNAs have a 5′-terminal m7G cap structure that is recognized by the eIF4E (cap-binding) subunit of eIF4F. Mammalian eIF4F also contains eIF4G and eIF4A subunits. eIF4A is an RNA-dependent ATPase and RNA helicase. After binding of eIF4F to the 5′ end of an mRNA, eIF4A and eIF4B melt the RNA structure in its 5′-nontranslated region, which facilitates binding of the 43S complex to the 5′ end of the mRNA. Ribosomal binding is thought to be mediated through interactions of eIF4G and eIF4B with ribosome-bound eIF3 (22, 29). The ribosomal complex then scans to the initiating AUG codon (35). Finally, eIF5 and eIF5B mediate the displacement of factors from the 40S subunit and joining of the 60S subunit to form an active 80S ribosome (40). eIF4G is a large adapter protein with a modular structure that plays a key coordinating role in the early stages of initiation (19, 31). Two related eIF4G proteins (eIF4GI and eIF4GII) encoded by two different genes exist in yeast and mammals (8, 9, 15, 19). Mammalian eIF4G can be divided into three distinct functional domains. The N-terminal third (amino acids [aa] 1 to 612) contains the eIF4E and PABP binding sites (15, 24); the middle third (aa 613 to 1090) binds eIF3, eIF4A, and RNA (3, 14, 22, 30, 41); and the C-terminal third (aa 1091 to 1560) contains a second eIF4A binding site (14, 22, 30) and a binding site for the protein kinase Mnk1 (43). eIF4G therefore acts as a platform for the assembly of a multiprotein-RNA complex to recruit the ribosome to a mRNA. Consistent with its central role in initiation, eIF4G is also an important target in the regulation of protein synthesis. The 4E binding proteins (4E-BPs) act as general inhibitors of cap-dependent translation by binding eIF4E and sequestering it from the rest of the eIF4F complex (6). Biochemical and structural studies have established that the 4E-BPs are molecular mimics of eIF4G and compete for the same binding site on the dorsal surface of eIF4E (11, 25). eIF4G is also a direct target for regulation by phosphorylation (44) and by proteolysis, both during apoptosis (26) and during infection by some picornaviruses such as poliovirus (10, 21, 22). Proteases encoded by these picornaviruses cleave eIF4G specifically, separating the eIF4E-PABP binding domain from the eIF4A and eIF3 binding sites. In contrast, other picornaviruses such as encephalomyocarditis virus (EMCV) inhibit cellular translation by dephosphorylating 4E-BP1 and thereby disrupting the eIF4E-eIF4G interaction (7). Both strategies abrogate the activity of eIF4F in initiation on capped mRNAs and thus lead to a shutoff of host cell translation. Initiation of translation of picornavirus mRNAs occurs by a noncanonical cap-independent mechanism of internal initiation that is mediated by a ∼400-nucleotide (nt) highly structured internal ribosomal entry site (IRES) that lies immediately upstream of the initiation codon (16). The EMCV IRES epitomizes those of a large group of picornaviruses, including all members of the Aphthovirus, Cardiovirus, and Parechovirus genera. We reconstituted EMCV IRES-mediated initiation in vitro using purified translation components and found that this process is ATP dependent and utilizes the same set of canonical eIFs as does cap-mediated initiation except for eIF1, eIF1A, eIF4E, PABP, and the amino-terminal third of eIF4G, to which the last two bind (37, 39, 40). The essential region of eIF4G corresponds to the carboxy-terminal proteolytic cleavage product that is generated during poliovirus infection. It binds specifically to the J-K domain of the IRES, in close proximity to the initiation codon, and recruits eIF4A and eIF4B to the IRES (20, 41). Its interaction with the IRES is essential for 48S complex formation, and its role in IRES-mediated initiation may be analogous to that of the eIF4E-eIF4G complex in initiation on capped mRNAs, i.e., recruiting factors and promoting ribosomal attachment at a defined location on the mRNA. The requirement for eIF4G and its specific binding to the J-K domain is a general characteristic of the mechanism of initiation on all EMCV-like picornavirus IRESs, such as those of foot-and-mouth disease virus, human parechovirus 1, and Theiler's murine encephalomyelitis virus (41a; V. G. Kolupaeva, unpublished data). In this article we define the minimum region of eIF4GI required for its specific interaction with the EMCV IRES and for support of 48S complex formation on this RNA. Our data provide evidence that the specific interaction of eIF4A with eIF4G significantly enhances its affinity for the IRES and indicate that the interaction of eIF4A and eIF4G is required to yield an active complex in IRES-mediated initiation.

Hepatitis C virus translation is initiated on a ∼330-nucleotide (nt)-long internal ribosomal entry site (IRES) at the 5′ end of the genome. In this process, a 43S preinitiation complex (comprising a 40S ribosomal subunit, eukaryotic initiation factor 3 (eIF3), and a ternary [eIF2-GTP-initiator tRNA] complex) binds the IRES in a precise manner so that the initiation codon is placed at the ribosomal P site. This binding step involves specific interactions between the IRES and different components of the 43S complex. The 40S subunit and eIF3 can bind to the IRES independently; previous analyses revealed that eIF3 binds specifically to an apical half of IRES domain III. Nucleotides in the IRES that are involved in the interaction with the 40S subunit were identified by RNase footprinting and mapped to the basal half of domain III and in domain IV. Interaction sites were identified in locations that have been found to be essential for IRES function, including (i) the apical loop residues GGG 266-268 in subdomain IIId and (ii) the pseudoknot. Extensive protection from RNase cleavage also occurred downstream of the pseudoknot in domain IV, flanking both sides of the initiation codon and corresponding in length to that of the mRNA-binding cleft of the 40S subunit. These results indicate that the 40S subunit makes multiple interactions with the IRES and suggest that only nucleotides in domain IV are inserted into the mRNA-binding cleft of the 40S subunit.

Initiation of eukaryotic protein synthesis begins with the ribosome separated into its 40S and 60S subunits. The 40S subunit first binds eukaryotic initiation factor (eIF) 3 and an eIF2-GTP-initiator transfer RNA ternary complex. The resulting complex requires eIF1, eIF1A, eIF4A, eIF4B and eIF4F to bind to a messenger RNA and to scan to the initiation codon. eIF5 stimulates hydrolysis of eIF2-bound GTP and eIF2 is released from the 48S complex formed at the initiation codon before it is joined by a 60S subunit to form an active 80S ribosome. Here we show that hydrolysis of eIF2-bound GTP induced by eIF5 in 48S complexes is necessary but not sufficient for the subunits to join. A second factor termed eIF5B (relative molecular mass 175,000) is essential for this process. It is a homologue of the prokaryotic initiation factor IF2 (re and, like it, mediates joining of subunits and has a ribosome-dependent GTPase activity that is essential for its function.

Initiation of translation on the bovine viral diarrhea virus (BVDV) internal ribosomal entry site (IRES) was reconstituted in vitro from purified translation components to the stage of 48S ribosomal initiation complex formation. Ribosomal binding and positioning on this mRNA to form a 48S complex did not require the initiation factors eIF4A, eIF4B, or eIF4F, and translation of this mRNA was resistant to inhibition by a trans-dominant eIF4A mutant that inhibited cap-mediated initiation of translation. The BVDV IRES contains elements that are bound independently by ribosomal 40S subunits and by eukaryotic initiation factor (eIF) 3, as well as determinants that mediate direct attachment of 43S ribosomal complexes to the initiation codon.

eIF1 is a universally conserved translation factor that is necessary for scanning and involved in initiation site selection. We have determined the solution structure of human eIF1 with an N-terminal His tag using NMR spectroscopy. Residues 29-113 of the native sequence form a tightly packed domain with two alpha-helices on one side of a five-stranded parallel and antiparallel beta-sheet. The fold is new but similar to that of several ribosomal proteins and RNA-binding domains. A likely binding site is indicated by yeast mutations and conserved residues located together on the surface. No interaction with recombinant eIF5 or the initiation site RNA GCCACAAUGGCA was detected by NMR, but GST pull-down experiments show that eIF1 binds specifically to the p110 subunit of eIF3. This interaction explains how eIF1 is recruited to the 40S ribosomal subunit.

This work was supported by grants from the NIH, the NSF and the Council for Tobacco Research.

The scanning model of translation initiation is a coherent description of how eukaryotic ribosomes reach the initiation codon after being recruited to the capped 5' end of messenger RNA. Five eukaryotic initiation factors (eIF 2, 3, 4A, 4B and 4F) with established functions have been assumed to be sufficient to mediate this process. Here we report that eIF1 and eIF1A are also both essential for translation initiation. In their absence, 43S ribosomal preinitiation complexes incubated with ATP, eIF4A, eIF4B and eIF4F bind exclusively to the cap-proximal region but are unable to reach the initiation codon. Individually, eIF1A enhances formation of this cap-proximal complex, and eIF1 weakly promotes formation of a 48S ribosomal complex at the initiation codon. These proteins act synergistically to mediate assembly of ribosomal initiation complexes at the initiation codon and dissociate aberrant complexes from the mRNA.