β-Hairpin Loop of Eukaryotic Initiation Factor 1 (eIF1) Mediates 40 S Ribosome Binding to Regulate Initiator tRNAMet Recruitment and Accuracy of AUG Selection in Vivo

Recognition of the translation initiation codon is thought to require dissociation of eIF1 from the 40 S ribosomal subunit, enabling irreversible GTP hydrolysis (P_i release) by the eIF2·GTP·Met-tRNA_i ternary complex (TC), rearrangement of the 40 S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNA_i to the P site. The crystal structure of a Tetrahymena 40 S·eIF1 complex revealed several basic amino acids in eIF1 contacting 18 S rRNA, and we tested the prediction that their counterparts in yeast eIF1 are required to prevent premature eIF1 dissociation from scanning ribosomes at non-AUG triplets. Supporting this idea, substituting Lys-60 in helix α1, or either Lys-37 or Arg-33 in β-hairpin loop-1, impairs binding of yeast eIF1 to 40 S·eIF1A complexes in vitro, and it confers increased initiation at UUG codons (Sui⁻ phenotype) or lethality, in a manner suppressed by overexpressing the mutant proteins or by an eIF1A mutation (17–21) known to impede eIF1 dissociation in vitro. The eIF1 Sui⁻ mutations also derepress translation of GCN4 mRNA, indicating impaired ternary complex loading, and this Gcd⁻ phenotype is likewise suppressed by eIF1 overexpression or the 17–21 mutation. These findings indicate that direct contacts of eIF1 with 18 S rRNA seen in the Tetrahymena 40 S·eIF1 complex are crucial in yeast to stabilize the open conformation of the 40 S subunit and are required for rapid TC loading and ribosomal scanning and to impede rearrangement to the closed complex at non-AUG codons. Finally, we implicate the unstructured N-terminal tail of eIF1 in blocking rearrangement to the closed conformation in the scanning preinitiation complex.     

Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

In eukaryotes, phosphorylation of translation initiation factor 2α (eIF2α) by the kinase Gcn2 (general control nonderepressible 2) is a key response to amino acid starvation. Sensing starvation requires that Gcn2 directly contacts its effector protein Gcn1, and both must contact the ribosome. We have proposed that Gcn2 is activated by uncharged tRNA bound to the ribosomal decoding (A) site, in a manner facilitated by ribosome-bound Gcn1. Protein synthesis requires cyclical association of eukaryotic elongation factors (eEFs) with the ribosome. Gcn1 and Gcn2 are large proteins, raising the question of whether translation and monitoring amino acid availability can occur on the same ribosome. Part of the ribosome-binding domain in Gcn1 has homology to one of the ribosome-binding domains in eEF3, suggesting that these proteins utilize overlapping binding sites on the ribosome and consequently cannot function simultaneously on the same ribosome. Supporting this idea, we found that eEF3 overexpression in Saccharomyces cerevisiae diminished growth on amino acid starvation medium (Gcn⁻ phenotype) and decreased eIF2α phosphorylation, and that the growth defect associated with constitutively active Gcn2 was diminished by eEF3 overexpression. Overexpression of the eEF3 HEAT domain, or C terminus, was sufficient to confer a Gcn⁻ phenotype, and both fragments have ribosome affinity. eEF3 overexpression did not significantly affect Gcn1-ribosome association, but it exacerbated the Gcn⁻ phenotype of Gcn1-M7A that has reduced ribosome affinity. Together, this suggests that eEF3 blocks Gcn1 regulatory function on the ribosome. We propose that the Gcn1-Gcn2 complex only functions on ribosomes with A-site-bound uncharged tRNA, because eEF3 does not occupy these stalled complexes.     

Evidence for a Negative Cooperativity between eIF5A and eEF2 on Binding to the Ribosome

eIF5A is the only protein known to contain the essential and unique amino acid residue hypusine. eIF5A functions in both translation initiation due to its stimulation of methionyl-puromycin synthesis and translation elongation, being highly required for peptide-bound formation of specific ribosome stalling sequences such as poly-proline. The functional interaction between eIF5A, tRNA, and eEF2 on the surface of the ribosome is further clarified herein. Fluorescence anisotropy assays were performed to determine the affinity of eIF5A to different ribosomal complexes and reveal its interaction exclusively and directly with the 60S ribosomal subunit in a hypusine-dependent manner (K_i^{60S-eIF5A-Hyp} = 16 nM, K_i^{60S-eIF5A-Lys} = 385 nM). A 3-fold increase in eIF5A affinity to the 80S is observed upon charged-tRNA_i^Met binding, indicating positive cooperativity between P-site tRNA binding and eIF5A binding to the ribosome. Previously identified conditional mutants of yeast eIF5A, eIF5A^Q22H/L93F and eIF5A^K56A, display a significant decrease in ribosome binding affinity. Binding affinity between ribosome and eIF5A-wild type or mutants eIF5A^K56A, but not eIF5A^Q22H/L93F, is impaired in the presence of eEF2 by 4-fold, consistent with negative cooperativity between eEF2 and eIF5A binding to the ribosome. Interestingly, high-copy eEF2 is toxic only to eIF5A^Q22H/L93F and causes translation elongation defects in this mutant. These results suggest that binding of eEF2 to the ribosome alters its conformation, resulting in a weakened affinity of eIF5A and impairment of this interplay compromises cell growth due to translation elongation defects.     

Translation initiation complex formation in the crenarchaeon Sulfolobus solfataricus

The function of initiation factors in and the sequence of events during translation initiation have been intensively studied in Bacteria and Eukaryotes, whereas in Archaea knowledge on these functions/processes is limited. By employing chemical probing, we show that translation initiation factor aIF1 of the model crenarchaeon Sulfolobus solfataricus binds to the same area on the ribosome as the bacterial and eukaryal orthologs. Fluorescence energy transfer assays (FRET) showed that aIF1, like its eukaryotic and bacterial orthologs, has a fidelity function in translation initiation complex formation, and that both aIF1 and aIF1A exert a synergistic effect in stimulating ribosomal association of the Met-tRNAi^Met binding factor a/eIF2. However, as in Eukaryotes their effect on a/eIF2 binding appears to be indirect. Moreover, FRET was used to analyze for the first time the sequence of events toward translation initiation complex formation in an archaeal model system. These studies suggested that a/eIF2-GTP binds first to the ribosome and then recruits Met-tRNAi^Met, which appears to comply with the operational mode of bacterial IF2, and deviates from the shuttle function of the eukaryotic counterpart eIF2. Thus, despite the resemblance of eIF2 and a/eIF2, recruitment of initiator tRNA to the ribosome is mechanistically different in Pro- and Eukaryotes.     

Yeast 18 S rRNA Is Directly Involved in the Ribosomal Response to Stringent AUG Selection during Translation Initiation

In eukaryotes, the 40 S ribosomal subunit serves as the platform of initiation factor assembly, to place itself precisely on the AUG start codon. Structural arrangement of the 18 S rRNA determines the overall shape of the 40 S subunit. Here, we present genetic evaluation of yeast 18 S rRNA function using 10 point mutations altering the polysome profile. All the mutants reduce the abundance of the mutant 40 S, making it limiting for translation initiation. Two of the isolated mutations, G875A, altering the core of the platform domain that binds eIF1 and eIF2, and A1193U, changing the h31 loop located below the P-site tRNA_i^Met, show phenotypes indicating defective regulation of AUG selection. Evidence is provided that these mutations reduce the interaction with the components of the preinitiation complex, thereby inhibiting its function at different steps. These results indicate that the 18 S rRNA mutations impair the integrity of scanning-competent preinitiation complex, thereby altering the 40 S subunit response to stringent AUG selection. Interestingly, nine of the mutations alter the body/platform domains of 18 S rRNA, potentially affecting the bridges to the 60 S subunit, but they do not change the level of 18 S rRNA intermediates. Based on these results, we also discuss the mechanism of the selective degradation of the mutant 40 S subunits.     

Factor requirements for translation initiation on the Simian picornavirus internal ribosomal entry site

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.     

The eIF2A knockout mouse

Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the early 1970s as a factor capable of stimulating initiator methionyl-tRNAi (Met-tRNA^Met_i) binding to 40S ribosomal subunits in vitro. However, in contrast to the eIF2, which stimulates Met-tRNA^Met_i binding to 40S ribosomal subunits in a GTP-dependent manner, eIF2A didn't reveal any GTP-dependence, but instead was found to direct binding of the Met-tRNA^Met_i to 40S ribosomal subunits in a codon-dependent manner. eIF2A appears to be highly conserved across eukaryotic species, suggesting conservation of function in evolution. The yeast Saccharomyces cerevisae eIF2A null mutant revealed no apparent phenotype, however, it was found that in yeast eIF2A functions as a suppressor of internal ribosome entry site (IRES)-mediated translation. It was thus suggested that eIF2A my act by impinging on the expression of specific mRNAs. Subsequent studies in mammalian cell systems implicated eIF2A in non-canonical (non-AUG-dependent) translation initiation events involving near cognate UUG and CUG codons. Yet, the role of eIF2A in cellular functions remains largely enigmatic. As a first step toward characterization of the eIF2A function in mammalian systems in vivo, we have obtained homozygous eIF2A-total knockout (KO) mice, in which a gene trap cassette was inserted between eIF2A exons 1 and 2 disrupting expression of all exons downstream of the insertion. The KO mice strain is viable and to date displays no apparent phenotype. We believe that the eIF2A KO mice strain will serve as a valuable tool for researchers studying non-canonical initiation of translation in vivo.     

AIMP3/p18 Controls Translational Initiation by Mediating the Delivery of Charged Initiator tRNA to Initiation Complex

Aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMPs) are nonenzymatic scaffolding proteins that comprise multisynthetase complex (MSC) with nine aminoacyl-tRNA synthetases in higher eukaryotes. Among the three AIMPs, AIMP3/p18 is strongly anchored to methionyl-tRNA synthetase (MRS) in the MSC. MRS attaches methionine (Met) to initiator tRNA (tRNA_i^Met) and plays an important role in translation initiation. It is known that AIMP3 is dispatched to nucleus or nuclear membrane to induce DNA damage response or senescence; however, the role of AIMP3 in translation as a component of MSC and the meaning of its interaction with MRS are still unclear. Herein, we observed that AIMP3 specifically interacted with Met-tRNA_i^Metin vitro, while it showed little or reduced interaction with unacylated or lysine-charged tRNA_i^Met. In addition, AIMP3 discriminates Met-tRNA_i^Met from Met-charged elongator tRNA based on filter-binding assay. Pull‐down assay revealed that AIMP3 and MRS had noncompetitive interaction with eukaryotic initiation factor 2 (eIF2) γ subunit (eIF2γ), which is in charge of binding with Met-tRNA_i^Met for the delivery of Met-tRNA_i^Met to ribosome. AIMP3 recruited active eIF2γ to the MRS-AIMP3 complex, and the level of Met-tRNA_i^Met bound to eIF2 complex was reduced by AIMP3 knockdown resulting in reduced protein synthesis. All these results suggested the novel function of AIMP3 as a critical mediator of Met-tRNA_i^Met transfer from MRS to eIF2 complex for the accurate and efficient translation initiation.     

Enhanced eIF1 binding to the 40S ribosome impedes conformational rearrangements of the preinitiation complex and elevates initiation accuracy

In the current model of translation initiation by the scanning mechanism, eIF1 promotes an open conformation of the 40S subunit competent for rapidly loading the eIF2·GTP·Met-tRNA_i ternary complex (TC) in a metastable conformation (P_OUT) capable of sampling triplets entering the P site while blocking accommodation of Met-tRNA_i in the P_IN state and preventing completion of GTP hydrolysis (P_i release) by the TC. All of these functions should be reversed by eIF1 dissociation from the preinitiation complex (PIC) on AUG recognition. We tested this model by selecting eIF1 Ssu⁻ mutations that suppress the elevated UUG initiation and reduced rate of TC loading in vivo conferred by an eIF1 (Sui⁻) substitution that eliminates a direct contact of eIF1 with the 40S subunit. Importantly, several Ssu⁻ substitutions increase eIF1 affinity for 40S subunits in vitro, and the strongest-binding variant (D61G), predicted to eliminate ionic repulsion with 18S rRNA, both reduces the rate of eIF1 dissociation and destabilizes the P_IN state of TC binding in reconstituted PICs harboring Sui⁻ variants of eIF5 or eIF2. These findings establish that eIF1 dissociation from the 40S subunit is required for the P_IN mode of TC binding and AUG recognition and that increasing eIF1 affinity for the 40S subunit increases initiation accuracy in vivo. Our results further demonstrate that the GTPase-activating protein eIF5 and β-subunit of eIF2 promote accuracy by controlling eIF1 dissociation and the stability of TC binding to the PIC, beyond their roles in regulating GTP hydrolysis by eIF2.     

Translation initiation factors are not required for Dicistroviridae IRES function in vivo

The cricket paralysis virus (CrPV) intergenic region (IGR) internal ribosome entry site (IRES) uses an unusual mechanism of initiating translation, whereby the IRES occupies the P-site of the ribosome and the initiating tRNA enters the A-site. In vitro experiments have demonstrated that the CrPV IGR IRES is able to bind purified ribosomes and form 80S complexes capable of synthesizing small peptides in the absence of any translation initiation factors. These results suggest that initiation by this IRES is factor-independent. To determine whether the IGR IRES functions in the absence of initiation factors in vivo, we assayed IGR IRES activity in various yeast strains harboring mutations in canonical translation initiation factors. We used a dicistronic reporter assay in yeast to determine whether the CrPV IGR IRES is able to promote translation sufficient to support growth in the presence of various deletions or mutations in translation initiation factors. Using this assay, we have previously shown that the CrPV IGR IRES functions efficiently in yeast when ternary complexes (eIF2•GTP•initiator tRNA^met) are reduced. Here, we demonstrate that the CrPV IGR IRES activity does not require the eukaryotic initiation factors eIF4G1 or eIF5B, and it is enhanced when eIF2B, the eIF3b subunit of eIF3, or eIF4E are impaired. Taken together, these data support a model in which the CrPV IGR IRES is capable of initiating protein synthesis in the absence of any initiation factors in vivo, and suggests that the CrPV IGR IRES initiates translation by directly recruiting the ribosomal subunits in vivo.     

Human Eukaryotic Initiation Factor 4G (eIF4G) Protein Binds to eIF3c, -d,and -e to Promote mRNA Recruitment to the Ribosome

Recruitment of mRNA to the 40S ribosomal subunit requires the coordinated interaction of a large number of translation initiation factors. In mammals, the direct interaction between eukaryotic initiation factor 4G (eIF4G) and eIF3 is thought to act as the molecular bridge between the mRNA cap-binding complex and the 40S subunit. A discrete ∼90 amino acid domain in eIF4G is responsible for binding to eIF3, but the identity of the eIF3 subunit(s) involved is less clear. The eIF3e subunit has been shown to directly bind eIF4G, but the potential role of other eIF3 subunits in stabilizing this interaction has not been investigated. It is also not clear if the eIF4A helicase plays a role in stabilizing the interaction between eIF4G and eIF3. Here, we have used a fluorescence anisotropy assay to demonstrate that eIF4G binds to eIF3 independently of eIF4A binding to the middle region of eIF4G. By using a site-specific cross-linking approach, we unexpectedly show that the eIF4G-binding surface in eIF3 is comprised of the -c, -d and -e subunits. Screening multiple cross-linker positions reveals that eIF4G contains two distinct eIF3-binding subdomains within the previously identified eIF3-binding domain. Finally, by employing an eIF4G-dependent translation assay, we establish that both of these subdomains are required for efficient mRNA recruitment to the ribosome and stimulate translation. Our study reveals unexpected complexity to the eIF3-eIF4G interaction that provides new insight into the regulation of mRNA recruitment to the human ribosome.     

Translation initiation by factor-independent binding of eukaryotic ribosomes to internal ribosomal entry sites

Two exceptional mechanisms of eukaryotic translation initiation have recently been identified that differ fundamentally from the canonical factor-mediated, end-dependent mechanism of ribosomal attachment to mRNA. Instead, ribosomal 40S subunits bind in a factor-independent manner to the internal ribosomal entry site (IRES) in an mRNA. These two mechanisms are exemplified by initiation on the unrelated approximately 300 nt.-long Hepatitis C virus (HCV) IRES and the approximately 200 nt.-long cricket paralysis virus (CrPV) intergenic region (IGR) IRES, respectively. Ribosomal binding involves interaction with multiple non-contiguous sites on these IRESs, and therefore also differs from the factor-independent attachment of prokaryotic ribosomes to mRNA, which involves base-pairing to the linear Shine-Dalgarno sequence. The HCV IRES binds to the solvent side of the 40S subunit, docks a domain of the IRES into the ribosomal exit (E) site and places the initiation codon in the ribosomal peptidyl (P) site. Subsequent binding of eIF3 and the eIF2-GTP/initiator tRNA complex to form a 48S complex is followed by subunit joining to form an 80S ribosome. The CrPV IRES binds to ribosomes in a very different manner, by occupying the ribosomal E and P sites in the intersubunit cavity, thereby excluding initiator tRNA. Ribosomes enter the elongation stage of translation directly, without any involvement of initiator tRNA or initiation factors, following recruitment of aminoacyl-tRNA to the ribosomal aminoacyl (A) site and translocation of it to the P site.     

eIF2-dependent and eIF2-independent modes of initiation on the CSFV IRES: a common role of domain II

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(iMet) to bind directly to the initiation codon, yielding 48S initiation complexes. We report that eIF5B or eIF5B/eIF3 also promote Met-tRNA(iMet) 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(iMet) 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.     

Thermodynamic and Kinetic Framework of Selenocysteyl-tRNASec Recognition by Elongation Factor SelB

SelB is a specialized translation elongation factor that delivers selenocysteyl-tRNA^Sec (Sec-tRNA^Sec) to the ribosome. Here we show that Sec-tRNA^Sec binds to SelB·GTP with an extraordinary high affinity (K_d = 0.2 pm). The tight binding is driven enthalpically and involves the net formation of four ion pairs, three of which may involve the Sec residue. The dissociation of tRNA from the ternary complex SelB·GTP·Sec-tRNA^Sec is very slow (0.3 h⁻¹), and GTP hydrolysis accelerates the release of Sec-tRNA^Sec by more than a million-fold (to 240 s⁻¹). The affinities of Sec-tRNA^Sec to SelB in the GDP or apoforms, or Ser-tRNA^Sec and tRNA^Sec to SelB in any form, are similar (K_d = 0.5 μm). Thermodynamic coupling in binding of Sec-tRNA^Sec and GTP to SelB ensures at the same time the specificity of Sec- versus Ser-tRNA^Sec selection and rapid release of Sec-tRNA^Sec from SelB after GTP cleavage on the ribosome. SelB provides an example for the evolution of a highly specialized protein-RNA complex toward recognition of unique set of identity elements. The mode of tRNA recognition by SelB is reminiscent of another specialized factor, eIF2, rather than of EF-Tu, the common delivery factor for all other aminoacyl-tRNAs, in line with a common evolutionary ancestry of SelB and eIF2.     

A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits

Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.     

eIF3j is located in the decoding center of the human 40S ribosomal subunit

Protein synthesis in all cells begins with the ordered binding of the small ribosomal subunit to messenger RNA (mRNA) and transfer RNA (tRNA). In eukaryotes, translation initiation factor 3 (eIF3) is thought to play an essential role in this process by influencing mRNA and tRNA binding through indirect interactions on the backside of the 40S subunit. Here we show by directed hydroxyl radical probing that the human eIF3 subunit eIF3j binds to the aminoacyl (A) site and mRNA entry channel of the 40S subunit, placing eIF3j directly in the ribosomal decoding center. eIF3j also interacts with eIF1A and reduces 40S subunit affinity for mRNA. A high affinity for mRNA is restored upon recruitment of initiator tRNA, even though eIF3j remains in the mRNA-binding cleft in the presence of tRNA. These results suggest that eIF3j functions in part by regulating access of the mRNA-binding cleft in response to initiation factor binding.     

Translation Initiation Requires Cell Division Cycle 123 (Cdc123) to Facilitate Biogenesis of the Eukaryotic Initiation Factor 2 (eIF2)

The eukaryotic translation initiation factor 2 (eIF2) is central to the onset of protein synthesis and its modulation in response to physiological demands. eIF2, a heterotrimeric G-protein, is activated by guanine nucleotide exchange to deliver the initiator methionyl-tRNA to the ribosome. Here we report that assembly of the eIF2 complex in vivo depends on Cdc123, a cell proliferation protein conserved among eukaryotes. Mutations of CDC123 in budding yeast reduced the association of eIF2 subunits, diminished polysome levels, and increased GCN4 expression indicating that Cdc123 is critical for eIF2 activity. Cdc123 bound the unassembled eIF2γ subunit, but not the eIF2 complex, and the C-terminal domain III region of eIF2γ was both necessary and sufficient for Cdc123 binding. Alterations of the binding site revealed a strict correlation between Cdc123 binding, the biological function of eIF2γ, and its ability to assemble with eIF2α and eIF2β. Interestingly, high levels of Cdc123 neutralized the assembly defect and restored the biological function of an eIF2γ mutant. Moreover, the combined overexpression of eIF2 subunits rescued an otherwise inviable cdc123 deletion mutant. Thus, Cdc123 is a specific eIF2 assembly factor indispensable for the onset of protein synthesis. Human Cdc123 is encoded by a disease risk locus, and, therefore, eIF2 biogenesis control by Cdc123 may prove relevant for normal cell physiology and human health. This work identifies a novel step in the eukaryotic translation initiation pathway and assigns a biochemical function to a protein that is essential for growth and viability of eukaryotic cells.     

eIF2A mediates translation of hepatitis C viral mRNA under stress conditions

Translation of most mRNAs is suppressed under stress conditions. Phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF2), which delivers initiator tRNA (Met-tRNA(i)) to the P site of the 40S ribosomal subunit, is responsible for such translational suppression. However, translation of hepatitis C viral (HCV) mRNA is refractory to the inhibitory effects of eIF2α phosphorylation, which prevents translation by disrupting formation of the eIF2-GTP-Met-tRNA(i) ternary complex. Here, we report that eIF2A, an alternative initiator tRNA-binding protein, has a key role in the translation of HCV mRNA during HCV infection, in turn promoting eIF2α phosphorylation by activating the eIF2α kinase PKR. Direct interaction of eIF2A with the IIId domain of the HCV internal ribosome entry site (IRES) is required for eIF2A-dependent translation. These data indicate that stress-independent translation of HCV mRNA occurs by recruitment of eIF2A to the HCV IRES via direct interaction with the IIId domain and subsequent loading of Met-tRNA(i) to the P site of the 40S ribosomal subunit.     

Yeast initiator tRNA identity elements cooperate to influence multiple steps of translation initiation

All three kingdoms of life employ two methionine tRNAs, one for translation initiation and the other for insertion of methionines at internal positions within growing polypeptide chains. We have used a reconstituted yeast translation initiation system to explore the interactions of the initiator tRNA with the translation initiation machinery. Our data indicate that in addition to its previously characterized role in binding of the initiator tRNA to eukaryotic initiation factor 2 (eIF2), the initiator-specific A1:U72 base pair at the top of the acceptor stem is important for the binding of the eIF2.GTP.Met-tRNA(i) ternary complex to the 40S ribosomal subunit. We have also shown that the initiator-specific G:C base pairs in the anticodon stem of the initiator tRNA are required for the strong thermodynamic coupling between binding of the ternary complex and mRNA to the ribosome. This coupling reflects interactions that occur within the complex upon recognition of the start codon, suggesting that these initiator-specific G:C pairs influence this step. The effect of these anticodon stem identity elements is influenced by bases in the T loop of the tRNA, suggesting that conformational coupling between the D-loop-T-loop substructure and the anticodon stem of the initiator tRNA may occur during AUG codon selection in the ribosomal P-site, similar to the conformational coupling that occurs in A-site tRNAs engaged in mRNA decoding during the elongation phase of protein synthesis.