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SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on beta-globin, EMCV IRES and CrPV IRES mRNAs that use unrelated initiation mechanisms. In all instances, cleavage required Nsp1 and only canonical translational components (40S subunits and initiation factors), arguing against involvement of a putative cellular RNA endonuclease. Requirements for initiation factors differed for these mRNAs, reflecting their requirements for ribosomal attachment. Cleavage of CrPV IRES mRNA was supported by a minimal set of components consisting of 40S subunits and eIF3g's RRM domain. The cleavage site was located in the coding region 18 nucleotides downstream from the mRNA entrance indicating that cleavage occurs on the solvent side of the 40S subunit. Mutational analysis identified a positively charged surface on Nsp1's N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3g's RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of Nsp1-NTD and eIF3g's RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment.

In contrast to members of Picornaviridae which have long 5′-untranslated regions (5′UTRs) containing internal ribosomal entry sites (IRESs) that form five distinct classes, members of Caliciviridae typically have short 5′UTRs and initiation of translation on them is mediated by interaction of the viral 5′-terminal genome-linked protein (VPg) with subunits of eIF4F rather than by an IRES. The recent description of calicivirus genomes with 500–900nt long 5′UTRs was therefore unexpected and prompted us to examine them in detail. Sequence analysis and structural modelling of the atypically long 5′UTRs of Caliciviridae sp. isolate yc-13 and six other caliciviruses suggested that they contain picornavirus-like type 2 IRESs, whereas ruddy turnstone calicivirus (RTCV) and Caliciviridae sp. isolate hwf182cal1 calicivirus contain type 4 and type 5 IRESs, respectively. The suggestion that initiation on RTCV mRNA occurs by the type 4 IRES mechanism was confirmed experimentally using in vitro reconstitution. The high sequence identity between identified calicivirus IRESs and specific picornavirus IRESs suggests a common evolutionary origin. These calicivirus IRESs occur in a single phylogenetic branch of Caliciviridae and were likely acquired by horizontal gene transfer.

Translation initiation on structured mammalian mRNAs requires DHX29, a DExH protein that comprises a unique 534-aa-long N-terminal region (NTR) and a common catalytic DExH core. DHX29 binds to 40S subunits and possesses 40S-stimulated NTPase activity essential for its function. In the cryo-EM structure of DHX29-bound 43S preinitiation complexes, the main DHX29 density resides around the tip of helix 16 of 18S rRNA, from which it extends through a linker to the subunit interface forming an intersubunit domain next to the eIF1A binding site. Although a DExH core model can be fitted to the main density, the correlation between the remaining density and the NTR is unknown. Here, we present a model of 40S-bound DHX29, supported by directed hydroxyl radical cleavage data, showing that the intersubunit domain comprises a dsRNA-binding domain (dsRBD, aa 377–448) whereas linker corresponds to the long α-helix (aa 460–512) that follows the dsRBD. We also demonstrate that the N-terminal α-helix and the following UBA-like domain form a four-helix bundle (aa 90–166) that constitutes a previously unassigned section of the main density and resides between DHX29’s C-terminal α-helix and the linker. In vitro reconstitution experiments revealed the critical and specific roles of these NTR elements for DHX29’s function.

Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Biochemical studies revealed that direct binding of the IRES to the 40S ribosomal subunit places the initiation codon into the P site, where it base pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation-competent 80S ribosome. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. However, the structures of initiation complexes assembled on the HCV IRES, the transitions between different states, and the accompanying conformational changes have remained unknown. To fill these gaps, we now obtained cryo-EM structures of IRES initiation complexes, at resolutions up to 3.5 Å, that cover all major stages from the initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role of IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes and prepare them for subunit joining.

Initiation of translation on many viral mRNAs occurs by non-canonical mechanisms that involve 5’ end-independent binding of ribosomes to an internal ribosome entry site (IRES). The ~190 nt-long intergenic region (IGR) IRES of dicistroviruses such as Cricket paralysis virus (CrPV) initiates translation without Met-tRNAiMetor initiation factors. Advances in metagenomics have revealed numerous dicistrovirus-like genomes with shorter, structurally distinct IGRs, such as Nedicistrovirus (NediV) and Antarctic picorna-like virus 1 (APLV1). Like canonical IGR IRESs, the ~165 nt-long NediV-like IGRs comprise three domains, but they lack key canonical motifs, including L1.1a/L1.1b loops (which bind to the L1 stalk of the ribosomal 60S subunit) and the apex of stemloop V (SLV) (which binds to the head of the 40S subunit). Domain 2 consists of a compact, highly conserved pseudoknot (PKIII) from which a CrPV-like stemloop SLIV and an adjacent conserved 5nt-long stemloop protrude.In vitroreconstitution experiments showed that NediV-like IRESs initiate translation from a non-AUG codon and form elongation-competent 80S ribosomal complexes in the absence of initiation factors and Met-tRNAiMet. Unlike canonical IGR IRESs, NediV-like IRESs bind directly to the peptidyl (P) site of ribosomes leaving the aminoacyl (A) site accessible for decoding. The related structures of NediV-like IRESs and their common mechanism of action indicate that they exemplify a distinct class of IGR IRES.

SUMMARYHepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Direct binding of the IRES to the 40S subunit places the initiation codon into the P site, where it base-pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Then, eIF5 and eIF5B mediate subunit joining. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. Here, we present cryo-EM structures of IRES initiation complexes at resolutions up to 3.5 Å that cover all major stages from initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role for IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes that prepare them for subunit joining.

Giant viruses have extraordinarily large dsDNA genomes, and exceptionally, they encode various components of the translation apparatus, including tRNAs, aminoacyl-tRNA synthetases and translation factors. Here, we focused on the elongation factor 1 (EF1) family of viral translational GTPases (trGTPases), using computational and functional approaches to shed light on their functions. Multiple sequence alignment indicated that these trGTPases clustered into two groups epitomized by members of Mimiviridae and Marseilleviridae, respectively. trGTPases in the first group were more closely related to GTP-binding protein 1 (GTPBP1), whereas trGTPases in the second group were closer to eEF1A, eRF3 and Hbs1. Functional characterization of representative GTPBP1-like trGTPases (encoded by Hirudovirus, Catovirus and Moumouvirus) using in vitro reconstitution revealed that they possess eEF1A-like activity and can deliver cognate aa-tRNAs to the ribosomal A site during translation elongation. By contrast, representative eEF1A/eRF3/Hbs1-like viral trGTPases, encoded by Marseillevirus and Lausannevirus, have eRF3-like termination activity and stimulate peptide release by eRF1. Our analysis identified specific aspects of the functioning of these viral trGTPases with eRF1 of human, amoebal and Marseillevirus origin.

Members of Picornaviridae and of the Hepacivirus, Pegivirus and Pestivirus genera of Flaviviridae all contain an internal ribosomal entry site (IRES) in the 5′-untranslated region (5′UTR) of their genomes. Each class of IRES has a conserved structure and promotes 5′-end-independent initiation of translation by a different mechanism. Picornavirus 5′UTRs, including the IRES, evolve independently of other parts of the genome and can move between genomes, most commonly by intratypic recombination. We review accumulating evidence that IRESs are genetic entities that can also move between members of different genera and even between families. Type IV IRESs, first identified in the Hepacivirus genus, have subsequently been identified in over 25 genera of Picornaviridae, juxtaposed against diverse coding sequences. In several genera, members have either type IV IRES or an IRES of type I, II or III. Similarly, in the genus Pegivirus, members contain either a type IV IRES or an unrelated type; both classes of IRES also occur in members of the genus Hepacivirus. IRESs utilize different mechanisms, have different factor requirements and contain determinants of viral growth, pathogenesis and cell type specificity. Their dissemination between viruses by horizontal gene transfer has unexpectedly emerged as an important facet of viral evolution.

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.

The RNA exosome is a multisubunit protein complex that exhibits a 3' to 5' exoribonuclease activity, endoribonuclease activity, and participates in a variety of RNA processing and degradation pathways in both the nucleus and the cytoplasm. Exosomes interact with various cofactors which target them to specific RNA substrates and processes. Investigation of the mechanisms by which mammalian RNA exosomes are targeted to specific RNA substrates requires the development of in vitro approaches for purification of exosomes and their co-factors, assembly of substrates and monitoring of the exosomal activity. Here, we describe protocols for in vitro reconstitution of ribosomal 80S elongation complexes on cap-labeled mRNAs and for assaying exosomal degradation of mRNAs in such complexes depending on the presence of GTPBP1, which has previously been implicated in directing the exosome to mRNA targets.

The RNA exosome is a multisubunit protein complex that exhibits a 3' to 5' exoribonuclease activity, endoribonuclease activity, and participates in a variety of RNA processing and degradation pathways in both the nucleus and the cytoplasm. Exosomes interact with various cofactors which target them to specific RNA substrates and processes. Investigation of the mechanisms by which mammalian RNA exosomes are targeted to specific RNA substrates requires the development of in vitro approaches for purification of exosomes and their co-factors, assembly of substrates and monitoring of the exosomal activity. Here, we describe protocols for in vitro reconstitution of ribosomal 80S elongation complexes on cap-labeled mRNAs and for assaying exosomal degradation of mRNAs in such complexes depending on the presence of GTPBP1, which has previously been implicated in directing the exosome to mRNA targets.

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.

Summary The evolutionarily conserved Ski2-Ski3-Ski8 (Ski) complex containing the 3′→5′ RNA helicase Ski2 binds to 80S ribosomes near the mRNA entrance and facilitates 3′→5′ exosomal degradation of mRNA during ribosome-associated mRNA surveillance pathways. Here, we assayed Ski’s activity using an in vitro reconstituted translation system and report that this complex efficiently extracts mRNA from 80S ribosomes in the 3′→5′ direction in a nucleotide-by-nucleotide manner. The process is ATP dependent and can occur on pre- and post-translocation ribosomal complexes. The Ski complex can engage productively with mRNA and extract it from 80S complexes containing as few as 19 (but not 13) 3′-terminal mRNA nucleotides starting from the P site. The mRNA-extracting activity of the Ski complex suggests that its role in mRNA quality control pathways is not limited to acceleration of exosomal degradation and could include clearance of stalled ribosomes from mRNA, poising mRNA for degradation and rendering stalled ribosomes recyclable by Pelota/Hbs1/ABCE1.

GTP-binding protein 1 (GTPBP1) and GTPBP2 comprise a divergent group of translational GTPases with obscure functions, which are most closely related to eEF1A, eRF3, and Hbs1. Although recent reports implicated GTPBPs in mRNA surveillance and ribosome-associated quality control, how they perform these functions remains unknown. Here, we demonstrate that GTPBP1 possesses eEF1A-like elongation activity, delivering cognate aminoacyl-transfer RNA (aa-tRNA) to the ribosomal A site in a GTP-dependent manner. It also stimulates exosomal degradation of mRNAs in elongation complexes. The kinetics of GTPBP1-mediated elongation argues against its functioning in elongation per se but supports involvement in mRNA surveillance. Thus, GTP hydrolysis by GTPBP1 is not followed by rapid peptide bond formation, suggesting that after hydrolysis, GTPBP1 retains aa-tRNA, delaying its accommodation in the A site. In physiological settings, this would cause ribosome stalling, enabling GTPBP1 to elicit quality control programs; e.g., by recruiting the exosome. GTPBP1 can also deliver deacylated tRNA to the A site, indicating that it might function via interaction with deacylated tRNA, which accumulates during stresses. Although GTPBP2's binding to GTP was stimulated by Phe-tRNAPhe, suggesting that its function might also involve interaction with aa-tRNA, GTPBP2 lacked elongation activity and did not stimulate exosomal degradation, indicating that GTPBP1 and GTPBP2 have different functions.

Cadicivirus (CDV) is unique amongst picornaviruses in having a dicistronic genome with internal ribosomal entry sites (IRESs) preceding both open reading frames. Here, we investigated initiation on the 5′-terminal IRES. We report that the 982-nt long 5′UTR comprises 12 domains (d1-d12), five of which (d8-d12, nts 341–950) constitute a divergent Type I IRES. It comprises central elements (the apex of d10, d11 and the following polypyrimidine tract) that are homologous to corresponding elements in canonical Type 1 IRESs, and non-canonical flanking domains (d8, d9 and d12). In vitro reconstitution revealed that as with canonical Type I IRESs, 48S complex formation requires eukaryotic initiation factors (eIFs) 1, 1A, 2, 3, 4A, 4B and 4G, and the poly(C) binding protein 2 (PCBP2), and starts with specific binding of eIF4G/eIF4A to d11. However, in contrast to canonical Type I IRESs, subsequent recruitment of 43S ribosomal complexes does not require direct interaction of their eIF3 constituent with the IRES-bound eIF4G. On the other hand, the CDV IRES forms a 40S/eIF3/IRES ternary complex, with multiple points of contact. These additional interactions with translational components could potentially stimulate recruitment of the 43S complex and alleviate the necessity for direct eIF4G/eIF3 interaction.

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.

Summary Reinitiation is a strategy used by viruses to express several cistrons from one mRNA. Although extremely weak after translation of long open reading frames (ORFs) on cellular mRNAs, reinitiation occurs efficiently on subgenomic bicistronic calicivirus mRNAs, enabling synthesis of minor capsid proteins. The process is governed by a short element upstream of the restart AUG, designated "termination upstream ribosomal binding site" (TURBS). It contains the conserved Motif 1 complementary to h26 of 18S rRNA, displayed in the loop of a hairpin formed by species-specific Motifs 2/2 ∗ . To determine the advantages conferred on reinitiation by TURBS, we reconstituted this process in vitro on two model bicistronic calicivirus mRNAs. We found that post-termination ribosomal tethering of mRNA by TURBS allows reinitiation by post-termination 80S ribosomes and diminishes dependence on eukaryotic initiation factor 3 (eIF3) of reinitiation by recycled 40S subunits, which can be mediated either by eIFs 2/1/1A or by Ligatin following ABCE1-dependent or -independent splitting of post-termination complexes.

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.

Eukaryotic translation termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, we present a cryo-electron microscopy structure of pre-termination complexes associated with eRF1•eRF3•GDPNP at 9.7 -Å resolution, which corresponds to the initial pre-GTP hydrolysis stage of factor attachment and stop codon recognition. It reveals the ribosomal positions of eRFs and provides insights into the mechanisms of stop codon recognition and triggering of eRF3’s GTPase activity.

Eukaryotic translation termination results from the complex functional interplay between two eukaryotic release factors, eRF1 and eRF3, and the ribosome, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, using cryo-electron microscopy (cryo-EM) and flexible fitting, we determined the structure of eRF1–eRF3–guanosine 5′-[β,γ-imido]triphosphate (GMPPNP)-bound ribosomal pretermination complex (pre-TC), which corresponds to the initial, pre-GTP hydrolysis stage of factor attachment. Our results show that eukaryotic translation termination involves a network of interactions between the two release factors and the ribosome. Our structure provides mechanistic insight into the coordination between GTP hydrolysis by eRF3 and subsequent peptide release by eRF1.

Picornavirus mRNAs contain IRESs that sustain their translation during infection, when host protein synthesis is shut off. The major classes of picornavirus IRESs (Types 1 and 2) have distinct structures and sequences, but initiation on both is determined by their specific interaction with eIF4G. We report here that Aichivirus (AV), a member of the Kobuvirus genus of Picornaviridae, contains an IRES that differs structurally from Type 1 and Type 2 IRESs. Its function similarly involves interaction with eIF4G, but its eIF4G-interacting domain is structurally distinct, although it contains an apical eIF4G-interacting motif similar to that in Type 2 IRESs. Like Type 1 and Type 2 IRESs, AV IRES function is enhanced by pyrimidine tract-binding protein (PTB), but the pattern of PTB's interaction with each of these IRESs is distinct. Unlike all known IRESs, the AV IRES is absolutely dependent on DHX29, a requirement imposed by sequestration of its initiation codon in a stable hairpin.

No-go decay (NGD) and non-stop decay (NSD) are eukaryotic surveillance mechanisms that target mRNAs on which elongation complexes (ECs) are stalled by, for example, stable secondary structures (NGD) or due to the absence of a stop codon (NSD). Two interacting proteins Dom34(yeast)/Pelota(mammals) and Hbs1, which are paralogues of eRF1 and eRF3, are implicated in these processes. Dom34/Hbs1 were shown to promote dissociation of stalled ECs and release of intact peptidyl-tRNA. Using an in vitro reconstitution approach, we investigated the activities of mammalian Pelota/Hbs1 and report that Pelota/Hbs1 also induced dissociation of ECs and release of peptidyl-tRNA, but only in the presence of ABCE1. Whereas Pelota and ABCE1 were essential, Hbs1 had a stimulatory effect. Importantly, ABCE1/Pelota/Hbs1 dissociated ECs containing only a limited number of mRNA nucleotides downstream of the P-site, which suggests that ABCE1/Pelota/Hbs1 would disassemble NSD complexes stalled at the 3′-end, but not pre-cleavage NGD complexes stalled in the middle of mRNA. ABCE1/Pelota/Hbs1 also dissociated vacant 80S ribosomes, which stimulated 48S complex formation, suggesting that Pelota/Hbs1 have an additional role outside of NGD.

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.

Initiation codon selection in eukaryotes involves base-by-base inspection of the 5′-untranslated region of mRNA by scanning ribosomal 43S preinitiation complexes. We employed in vitro reconstitution to investigate factor requirements for this process and report that in the absence of eIF1 and DHX29, eIFs 4A, 4B and 4G promote efficient bypassing of stable stems by scanning 43S complexes and formation of 48S initiation complexes on AUG codons immediately upstream and downstream of such stems, without their unwinding. However, intact stems are not threaded through the entire mRNA Exit channel of the 40S subunit, resulting in incorrect positioning of mRNA upstream of the ribosomal P site in 48S complexes formed on AUG codons following intact stems, which renders them susceptible to dissociation by eIF1. In 48S complexes formed on AUG codons preceding intact stems, the stems are accommodated in the A site. Such aberrant complexes are destabilized by DHX29, which also ensures that mRNA enters the mRNA-binding cleft in a single-stranded form and therefore undergoes base-by-base inspection during scanning.

The ribosome-associated quality control (RQC) pathway degrades nascent chains (NCs) arising from interrupted translation. First, recycling factors split stalled ribosomes, yielding NC-tRNA/60S ribosome-nascent chain complexes (60S RNCs). 60S RNCs associate with NEMF, which recruits the E3 ubiquitin ligase Listerin that ubiquitinates NCs. The mechanism of subsequent ribosomal release of Ub-NCs remains obscure. We found that, in non-ubiquitinated 60S RNCs and 80S RNCs formed on non-stop mRNAs, tRNA is not firmly fixed in the P site, which allows peptidyl-tRNA hydrolase Ptrh1 to cleave NC-tRNA, suggesting the existence of a pathway involving release of non-ubiquitinated NCs. Association with NEMF and Listerin and ubiquitination of NCs results in accommodation of NC-tRNA, rendering 60S RNCs resistant to Ptrh1 but susceptible to ANKZF1, which induces specific cleavage in the tRNA acceptor arm, releasing proteasome-degradable Ub-NCs linked to four 3'-terminal tRNA nucleotides. We also found that TCF25, a poorly characterized RQC component, ensures preferential formation of the K48-ubiquitin linkage.

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.

The interaction between the poly(A)-binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA-binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB-1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB-1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP-dependent after the addition of YB-1. In this system, eIF4E binding to the cap structure is inhibited by YB-1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB-1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB-1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP.

Translational repressors, increasing evidence suggests, participate in the regulation of protein synthesis at the synapse, thus providing a basis for the long-term plastic modulation of synaptic strength. Dendritic BC1 RNA is a non-protein-coding RNA that represses translation at the level of initiation. However, the molecular mechanism of BC1 repression has remained unknown. Here we identify the catalytic activity of eukaryotic initiation factor 4A (eIF4A), an ATP-dependent RNA helicase, as a target of BC1-mediated translational control. BC1 RNA specifically blocks the RNA duplex unwinding activity of eIF4A but, at the same time, stimulates its ATPase activity. BC200 RNA, the primate-specific BC1 counterpart, targets eIF4A activity in identical fashion, as a result decoupling ATP hydrolysis from RNA duplex unwinding. In vivo, BC1 RNA represses translation of a reporter mRNA with 5' secondary structure. The eIF4A mechanism places BC RNAs in a central position to modulate protein synthesis in neurons.

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 Simian picornavirus type 9 (SPV9) 5′-untranslated region (5′ UTR) has been predicted to contain an internal ribosomal entry site (IRES) with structural elements that resemble domains of hepacivirus/pestivirus (HP) IRESs. In vitro reconstitution of initiation confirmed that this 5′ UTR contains an IRES and revealed that it has both functional similarities and differences compared to HP IRESs. Like HP IRESs, the SPV9 IRES bound directly to 40S subunits and eukaryotic initiation factor (eIF) 3, depended on the conserved domain IIId for ribosomal binding and consequently for function, and additionally required eIF2/initiator tRNA to yield 48S complexes that formed elongation-competent 80S ribosomes in the presence of eIF5, eIF5B, and 60S subunits. Toeprinting analysis revealed that eIF1A stabilized 48S complexes, whereas eIF1 induced conformational changes in the 40S subunit, likely corresponding to partial opening of the entry latch of the mRNA-binding channel, that were exacerbated by eIF3 and suppressed by eIF1A. The SPV9 IRES differed from HP IRESs in that its function was enhanced by eIF4A/eIF4F when the IRES was adjacent to the wild-type coding sequence, but was less affected by these factors or by a dominant negative eIF4A mutant when potentially less structured coding sequences were present. Exceptionally, this IRES promoted binding of initiator tRNA to the initiation codon in the P site of 40S subunits independently of eIF2. Although these 40S/IRES/tRNA complexes could not form active 80S ribosomes, this constitutes a second difference between the SPV9 and HP IRESs. eIF1 destabilized the eIF2-independent ribosomal binding of initiator tRNA.

SummaryAfter translational termination, mRNA and P site deacylated tRNA remain associated with ribosomes in posttermination complexes (post-TCs), which must therefore be recycled by releasing mRNA and deacylated tRNA and by dissociating ribosomes into subunits. Recycling of bacterial post-TCs requires elongation factor EF-G and a ribosome recycling factor RRF. Eukaryotes do not encode a RRF homolog, and their mechanism of ribosomal recycling is unknown. We investigated eukaryotic recycling using post-TCs assembled on a model mRNA encoding a tetrapeptide followed by a UAA stop codon and report that initiation factors eIF3, eIF1, eIF1A, and eIF3j, a loosely associated subunit of eIF3, can promote recycling of eukaryotic post-TCs. eIF3 is the principal factor that promotes splitting of posttermination ribosomes into 60S subunits and tRNA- and mRNA-bound 40S subunits. Its activity is enhanced by eIFs 3j, 1, and 1A. eIF1 also mediates release of P site tRNA, whereas eIF3j ensures subsequent dissociation of mRNA.

Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI ∼3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%–25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and −1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.

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 factor 1A stimulates 40S‐binding of the eukaryotic initiation factor 2 (eIF2)/GTP/Met‐tRNA i Met ternary complex (TC) and promotes scanning in vitro . eIF1A contains an OB‐fold present in bacterial IF1 plus N‐ and C‐terminal extensions. Truncating the C‐terminus (Δ C ) or mutating OB‐fold residues ( 66–70 ) of eIF1A reduced general translation in vivo but increased GCN4 translation (Gcd − phenotype) in a manner suppressed by overexpressing TC. Consistent with this, both mutations diminished 40S‐bound TC, eIF5 and eIF3 in vivo , and Δ C impaired TC recruitment in vitro . The assembly defects of the OB‐fold mutation can be attributed to reduced 40S‐binding of eIF1A, whereas Δ C impairs eIF1A function on the ribosome. A substitution in the C‐terminal helix ( 98 – 101 ) also reduced 43S assembly in vivo . Rather than producing a Gcd − phenotype, however, 98 – 101 impairs GCN4 derepression in a manner consistent with defective scanning by reinitiating ribosomes. Indeed, 98 – 101 allows formation of aberrant 48S complexes in vitro and increases utilization of non‐AUG codons in vivo . Thus, the OB‐fold is crucial for ribosome‐binding and the C‐terminal domain of eIF1A has eukaryotic‐specific functions in TC recruitment and scanning.

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.