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-tRNA i Met , 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. Protein synthesis begins following assembly of an initiation complex in which the initiation codon of an mRNA and the anticodon of initiator tRNA are base-paired in the ribosomal ''P'' site. There are similarities and significant differences in the mechanisms of initiation complex formation in prokaryotes and eukaryotes. A universal characteristic is that initiation starts with separated ribosomal subunits.In prokaryotes, the small (30S) ribosomal subunit binds mRNA and initiator tRNA in random order to form a complex that then undergoes conformational rearrangement, promoting codon-anticodon base-pairing at the P site and joining of the large (50S) subunit (Gualerzi and Pon 1990). Ribosome binding results from interactions between the 30S subunit and multiple recognition elements in the mRNA, such as the Shine-Dalgarno sequence (McCarthy and Brimacombe 1994). It does not depend on initiation factors. Ribosome-binding sites can occur at any position within an mRNA and as a result many prokaryotic mRNAs are polycistronic.In contrast, the small (40S) ribosomal subunit in eukaryotes requires several eukaryotic initiation factors (eIFs), first to bind initiator tRNA (as a ternary complex with eIF2 and GTP) to form a 43S complex and then to bind mRNA to form a 48S complex (Merrick 1992). The most common mechanism for recruitment of an mRNA is mediated by its capped 5Ј end, which is bound by the eIF4E subunit of eIF4F and then by the 43S complex. Ribosomal binding to mRNA and scanning to the initiation codon require ATP hydrolysis and involve eIF4A, eIF4B, and eIF4F. Most mRNAs that use this mechanism of ribosomal binding are monocistronic because initiation is usually limited to the 5Ј-most AUG codon.

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, eIF2͞GTP͞Met-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 eIF4A͞4G. 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.T ranslation of mRNA into protein begins after assembly of initiator tRNA (Met-tRNA i ), mRNA, and separated 40S and 60S ribosomal subunits into an 80S ribosome in which MettRNA i is positioned in the ribosomal P site at the initiation codon. The complex initiation process that leads to 80S ribosome formation consists of several linked stages that are mediated by eukaryotic initiation factors. These stages are:(i) Selection of initiator tRNA from the pool of elongator tRNAs by eukaryotic initiation factor (eIF)2 and binding of an eIF2͞GTP͞Met-tRNA i ternary complex and other eIFs to the 40S subunit to form a 43S preinitiation complex.(ii) Binding of the 43S complex to mRNA, which in most instances occurs by a mechanism that involves initial recognition of the m 7 G cap at the mRNA 5Ј-terminus by the eIF4E (cap-binding) subunit of eIF4F. Ribosomes bind to a subset of cellular and viral mRNAs as a result of cap-and endindependent internal ribosomal entry.(iii) Movement of the mRNA-bound ribosomal complex along the 5Ј nontranslated region (5ЈNTR) from its initial binding site to the initiation codon to form a 48S initiation complex in which the initiation codon is base paired to the anticodon of initiator tRNA.(iv) Displacement of factors from the 48S complex and joining of the 60S subunit to form an 80S ribosome, leaving Met-tRNA i in the ribosomal P site.Research in ...

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.Initiation of eukaryotic protein synthesis is the process of assembly of an 80S initiation complex containing initiator tRNA i Met , 40S, and 60S ribosomal subunits at the initiation codon of an mRNA (42). The first step in this process is formation of a 43S complex that consists of eukaryotic initiation factor 2 (eIF2), eIF3, and initiator Met-tRNA i Met bound to the 40S subunit. The second, rate-limiting step is the binding of mRNA to the 43S complex to form a 48S preinitiation complex. This occurs in one of two ways.The first mechanism is characteristic of most mRNAs. It involves recognition of the m 7 GpppX cap at the free 5Ј end of mRNA by the 4E subunit of eIF4F, followed by binding of the 43S complex at or close to the cap. Binding and 5Ј-3Ј scanning by this complex to the first AUG codon require ATP hydrolysis and unwinding of secondary structure in the 5Ј untranslated region (UTR) by eIF4F or eIF4A in cooperation with eIF4B. This mechanism of ribosomal entry imposes several restrictions on the structure of mRNAs that use it: they are capped, have relatively short, unstructured 5Ј UTRs, and are monocistronic because initiation is limited to the 5Ј-most AUG codon (34).A second, cap-independent mechanism of ribosome binding is used by mRNAs that contain an internal ribosomal entry site (IRES). IRES-mediated initiation is used by a number of cellular mRNAs, including those that encode the transcription factors TFIID (TATA-binding protein) and HAP4 (25), the growth factors fibroblast growth factor 2 and insulin-like growth factor 2 (63, 67), the homeotic genes Antennapedia and Ultrabithorax (45), the translation initiation factor eIF4G (14), and the immunoglobulin heavy-chain binding protein (39). This mechanism of translation initiation has been usurped by a number of viruses (e.g., references 49 and 65) and is exemplified by encephalomyocarditis virus (EMCV) (21, 26). The EMCV IRES is about 450 nucleotides (nt) long, is highly structured, and lies immediately upstream of AUG 834 , which is the 11th AUG triplet in the 5Ј UTR and the initiation codon for synthesis of the viral polyprotein (11, 27, 52, 66). The 5Ј UTRs of IRES-containing mRNAs differ from those of conventional mRNAs in many respects: they contain multiple AUG triplets and extensive secondary structure...

Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.DOI: http://dx.doi.org/10.7554/eLife.03971.001

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 first step in initiation of eukaryotic protein synthesis is formation of a 43S complex that consists of eukaryotic initiation factor 3 (eIF3) and a ternary complex (Met-tRNA i Met , eIF2, and GTP) bound to the 40S small ribosomal subunit (32). The second, rate-limiting step is binding of the 43S complex to mRNA. Translation of most mRNAs is cap dependent and follows binding of 43S complexes to the 5Ј m 7 G cap-proximal region. A smaller number of mRNAs contain an internal ribosomal entry site (IRES) within the 5Ј nontranslated region (5Ј NTR) that promotes binding of the 43S complex to a position in the mRNA that can be far downstream of the 5Ј terminus. Cap-dependent binding of ribosomes to eukaryotic mRNAs involves the initiation factors eIF4A, eIF4B, and eIF4F and ATP (48). We have recently found that the initiation mediated by the IRES of encephalomyocarditis virus (EMCV) has the same requirements (45).eIF4A exhibits RNA-dependent ATPase activity and, in conjunction with eIF4B, RNA helicase activity (29, 49). The RNA-binding activity of eIF4A is enhanced by eIF4B in an ATP-dependent manner (1). eIF4B is a 70-kDa phosphoprotein that contains an RNA recognition motif (RRM) near its amino terminus that can bind to 18S rRNA (34, 36) and a second centrally located domain that binds RNA in a sequence nonspecific manner (33, 38). eIF4F consists of eIF4E (capbinding protein), eIF4A, and eIF4G subunits. eIF4G is a 154-kDa polypeptide that coordinates the activity of eIF4F by interacting specifically with its eIF4A and eIF4E subunits, as well as with eIF3 (27, 31) and probably also with RNA (10). eIF4E binds to amino acids 409 to 457 of eIF4G (31). eIF4F also exhibits an RNA helicase activity that is stimulated by eIF4B and that i...

Unlike bacteria, a specialized eukaryotic initiation factor (eIF)-2, in the form of the ternary complex eIF2-GTP-Met-tRNA(i) (Met), is used to deliver the initiator tRNA to the ribosome in all eukaryotic cells. Here we show that the hepatitis C virus (HCV) internal ribosome entry site (IRES) can direct translation without eIF2 and its GTPase-activating protein eIF5. In addition to the general eIF2- and eIF5-dependent pathway of 80S complex assembly, the HCV IRES makes use of a bacterial-like pathway requiring as initiation factors only eIF5B (an analog of bacterial IF2) and eIF3. The switch from the conventional eukaryotic mode of translation initiation to the eIF2-independent mechanism occurs when eIF2 is inactivated by phosphorylation under stress conditions.

During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here, we report the tRNA binding to the P-site of 40 S ribosomes by a novel GTP-independent factor eIF2D isolated from mammalian cells. The binding of tRNA i Met occurs after the AUG codon finds its position in the P-site of 40 S ribosomes, the situation that takes place during initiation complex formation on the hepatitis C virus internal ribosome entry site or on some other specific RNAs (leaderless mRNA and A-rich mRNAs with relaxed scanning dependence). Its activity in tRNA binding with 40 S subunits does not require the presence of the aminoacyl moiety. Moreover, the factor possesses the unique ability to deliver non-Met (elongator) tRNAs into the P-site of the 40 S subunit. The corresponding gene is found in all eukaryotes and includes an SUI1 domain present also in translation initiation factor eIF1. The versatility of translation initiation strategies in eukaryotes is discussed.

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 ϳ450-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 IRES⅐eIF4G complexes did not further alter the pattern of chemical/enzymatic modification of the IRES.Initiation of protein synthesis in eukaryotes involves the sequential binding of small (40 S) and large (60 S) ribosomal subunits to an mRNA, leading to the assembly of an 80 S initiation complex at the initiation codon (1). The rate-limiting step in this process is recruitment of mRNAs to the 43 S preinitiation complex, which consists of the 40 S ribosomal subunit, methionine-initiator tRNA, and initiation factors, including eIF2 1 and eIF3. Ribosomal recruitment to most mRNAs requires the m 7 GpppN cap structure at the 5Ј end of the mRNA (2), but ribosomal binding to a smaller number of mRNAs is cap-and end-independent, and is instead mediated by an IRES in the 5Ј-untranslated region (3). One group of IRES elements is exemplified by encephalomyocarditis virus (EMCV) RNA (4). EMCV is a member of the cardiovirus genus of the Picornaviridae family.Eukaryotic initiation factor eIF4F, which consists of eIF4A, eIF4E, and eIF4G subunits, plays the central role in recruiting mRNAs to 43 S complexes during initiation. The cap structure is recognized by the 24-kDa cap-binding protein eIF4E. eIF4A is an RNA-dependent ATPase/RNA helicase that is thought to unwind cap-proximal regions of the 5Ј-untranslated region of an mRNA, permitting attachment of the 43 S complex (1, 2). The 154-kDa eIF4G subunit of eIF4F binds to these and other factors, thereby coordinating and enhancing their activities. eIF4E associates with amino acid residues 411-428 of eIF4G, eIF3 binds to the central part of eIF4G (residues 486 -886), and eIF4A binds to sites in the central and in the C-terminal thirds of eIF4G (5-7). eIF4G enhances binding both of eIF4E to the cap and of eIF4A to RNA (8, 9). The modular nature of eIF4G supports a model in which it acts as a bridge between the mRNA cap (via eIF4E) and the 40 S subunit (via eIF3, a constituent of the 43 S preinitiation complex) (6). In addition to containing ...