eIF5 and eIF5B together stimulate 48S initiation complex formation during ribosomal scanning

48S initiation complex (48S IC) formation is the first stage in the eukaryotic translation process. According to the canonical mechanism, 40S ribosomal subunit binds to the 5′-end of messenger RNA (mRNA) and scans its 5′-untranslated region (5′-UTR) to the initiation codon where it forms the 48S IC. Entire process is mediated by initiation factors. Here we show that eIF5 and eIF5B together stimulate 48S IC formation influencing initiation codon selection during ribosomal scanning. Initiation on non-optimal start codons—following structured 5′-UTRs, in bad AUG context, within few nucleotides from 5′-end of mRNA and CUG start codon—is the most affected. eIF5-induced hydrolysis of eIF2-bound GTP is essential for stimulation. GTP hydrolysis increases the probability that scanning ribosomal complexes will recognize and arrest scanning at a non-optimal initiation codon. Such 48S ICs are less stable owing to dissociation of eIF2*GDP from initiator tRNA, and eIF5B is then required to stabilize the initiator tRNA in the P site of 40S subunit. Alternative model that eIF5 and eIF5B cause 43S pre-initiation complex rearrangement favoring more efficient initiation codon recognition during ribosomal scanning is equally possible. Mutational analysis of eIF1A and eIF5B revealed distinct functions of eIF5B in 48S IC formation and subunit joining.     

Coordinated Movements of Eukaryotic Translation Initiation Factors eIF1, eIF1A,and eIF5 Trigger Phosphate Release from eIF2 in Response to Start Codon Recognition by the Ribosomal Preinitiation Complex

Accurate recognition of the start codon in an mRNA by the eukaryotic translation preinitiation complex (PIC) is essential for proper gene expression. The process is mediated by eukaryotic translation initiation factors (eIFs) in conjunction with the 40 S ribosomal subunit and (initiator) tRNA_i. Here, we provide evidence that the C-terminal tail (CTT) of eIF1A, which we previously implicated in start codon recognition, moves closer to the N-terminal domain of eIF5 when the PIC encounters an AUG codon. Importantly, this movement is coupled to dissociation of eIF1 from the PIC, a critical event in start codon recognition, and is dependent on the scanning enhancer elements in the eIF1A CTT. The data further indicate that eIF1 dissociation must be accompanied by the movement of the eIF1A CTT toward eIF5 in order to trigger release of phosphate from eIF2, which converts the latter to its GDP-bound state. Our results also suggest that release of eIF1 from the PIC and movement of the CTT of eIF1A are triggered by the same event, most likely accommodation of tRNA_i in the P site of the 40 S subunit driven by base pairing between the start codon in the mRNA and the anticodon in tRNA_i. Finally, we show that the C-terminal domain of eIF5 is responsible for the factor''s activity in antagonizing eIF1 binding to the PIC. Together, our data provide a more complete picture of the chain of molecular events that is triggered when the scanning PIC encounters an AUG start codon in the mRNA.     

Mammalian translation initiation factor eIF1 functions with eIF1A and eIF3 in the formation of a stable 40 S preinitiation complex

We have examined the role of the mammalian initiation factor eIF1 in the formation of the 40 S preinitiation complex using in vitro binding of initiator Met-tRNA (as Met-tRNA(i).eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA. We observed that, although both eIF1A and eIF3 are essential to generate a stable 40 S preinitiation complex, quantitative binding of the ternary complex to 40 S subunits also required eIF1. The 40 S preinitiation complex contained, in addition to eIF3, both eIF1 and eIF1A in a 1:1 stoichiometry with respect to the bound Met-tRNA(i). These three initiation factors also bind to free 40 S subunits, and the resulting complex can act as an acceptor of the ternary complex to form the 40 S preinitiation complex (40 S.eIF3.eIF1.eIF1A.Met-tRNA(i).eIF2.GTP). The stable association of eIF1 with 40 S subunits required the presence of eIF3. In contrast, the binding of eIF1A to free 40 S ribosomes as well as to the 40 S preinitiation complex was stabilized by the presence of both eIF1 and eIF3. These studies suggest that it is possible for eIF1 and eIF1A to bind the 40 S preinitiation complex prior to mRNA binding.     

A conformational change in the eukaryotic translation preinitiation complex and release of eIF1 signal recognition of the start codon

During eukaryotic translation initiation, ribosomal 43S complexes scan mRNAs for the correct AUG codon at which to begin translation. Start codon recognition triggers GTP hydrolysis, committing the complex to engagement at that point on the mRNA. While fidelity at this step is essential, the nature of the codon recognition event and the mechanism by which it activates GTP hydrolysis are poorly understood. Here we report the changes that occur within the 43S.mRNA complex in response to AUG codon recognition. eIF1 and eIF1A are key players in assembly of 43S.mRNA complexes capable of locating initiation codons. We observed FRET between these two factors when bound to the 40S subunit. Using steady-state FRET, anisotropy, and kinetic analyses, we demonstrate that start codon recognition results in a conformational change and release of eIF1 from the ribosome. These rearrangements probably play a role in triggering GTP hydrolysis and committing the complex to downstream events.     

eIF3: a versatile scaffold for translation initiation complexes

Translation initiation in eukaryotes depends on many eukaryotic initiation factors (eIFs) that stimulate both recruitment of the initiator tRNA, Met-tRNA(i)(Met), and mRNA to the 40S ribosomal subunit and subsequent scanning of the mRNA for the AUG start codon. The largest of these initiation factors, the eIF3 complex, organizes a web of interactions among several eIFs that assemble on the 40S subunit and participate in the different reactions involved in translation. Structural analysis suggests that eIF3 performs this scaffolding function by binding to the 40S subunit on its solvent-exposed surface rather than on its interface with the 60S subunit, where the decoding sites exist. This location of eIF3 seems ideally suited for its other proposed regulatory functions, including reinitiating translation on polycistronic mRNAs and acting as a receptor for protein kinases that control protein synthesis.     

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.     

Principles of start codon recognition in eukaryotic translation initiation

Selection of the correct start codon during initiation of translation on the ribosome is a key event in protein synthesis. In eukaryotic initiation, several factors have to function in concert to ensure that the initiator tRNA finds the cognate AUG start codon during mRNA scanning. The two initiation factors eIF1 and eIF1A are known to provide important functions for the initiation process and codon selection. Here, we have used molecular dynamics free energy calculations to evaluate the energetics of initiator tRNA binding to different near-cognate codons on the yeast 40S ribosomal subunit, in the presence and absence of these two initiation factors. The results show that eIF1 and eIF1A together cause a relatively uniform and high discrimination against near-cognate codons. This works such that eIF1 boosts the discrimination against a first position near-cognate G-U mismatch, and also against a second position A-A base pair, while eIF1A mainly acts on third codon position. The computer simulations further reveal the structural basis of the increased discriminatory effect caused by binding of eIF1 and eIF1A to the 40S ribosomal subunit.     

Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40 S ribosomal preinitiation complex.

We have used an in vitro translation initiation assay to investigate the requirements for the efficient transfer of Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA (or an AUG codon) to form the 40 S preinitiation complex. We observed that the 17-kDa initiation factor eIF1A is necessary and sufficient to mediate nearly quantitative transfer of Met-tRNAf to isolated 40 S ribosomal subunits. However, the addition of 60 S ribosomal subunits to the 40 S preinitiation complex formed under these conditions disrupted the 40 S complex resulting in dissociation of Met-tRNAf from the 40 S subunit. When the eIF1A-dependent preinitiation reaction was carried out with 40 S ribosomal subunits that had been preincubated with eIF3, the 40 S preinitiation complex formed included bound eIF3 (40 S.eIF3. Met-tRNAf.eIF2.GTP). In contrast to the complex lacking eIF3, this complex was not disrupted by the addition of 60 S ribosomal subunits. These results suggest that in vivo, both eIF1A and eIF3 are required to form a stable 40 S preinitiation complex, eIF1A catalyzing the transfer of Met-tRNAf.eIF2.GTP to 40 S subunits, and eIF3 stabilizing the resulting complex and preventing its disruption by 60 S ribosomal subunits.     

Functions of eIF3 downstream of 48S assembly impact AUG recognition and GCN4 translational control

The binding of eIF2-GTP-tRNA(i)(Met) ternary complex (TC) to 40S subunits is impaired in yeast prt1-1 (eIF3b) mutant extracts, but evidence is lacking that TC recruitment is a critical function of eIF3 in vivo. If TC binding was rate-limiting in prt1-1 cells, overexpressing TC should suppress the temperature-sensitive phenotype and GCN4 translation should be strongly derepressed in this mutant, but neither was observed. Rather, GCN4 translation is noninducible in prt1-1 cells, and genetic analysis indicates defective ribosomal scanning between the upstream open reading frames that mediate translational control. prt1-1 cells also show reduced utilization of a near-cognate start codon, implicating eIF3 in AUG selection. Using in vivo cross-linking, we observed accumulation of TC and mRNA/eIF4G on 40S subunits and a 48S 'halfmer' in prt1-1 cells. Genetic evidence suggests that 40S-60S subunit joining is not rate-limiting in the prt1-1 mutant. Thus, eIF3b functions between 48S assembly and subunit joining to influence AUG recognition and reinitiation on GCN4 mRNA. Other mutations that disrupt eIF2-eIF3 contacts in the multifactor complex (MFC) diminished 40S-bound TC, indicating that MFC formation enhances 43S assembly in vivo.     

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.     

Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing

The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNA_i^Met attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNA_i^Met, but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A''s OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNA_i^Met reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNA_i^Met, consistent with its suggested role in promoting the ‘closed’ conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the ‘closed’ complex and is likely ejected from the P-site upon start codon recognition.     

The Indispensable N-Terminal Half of eIF3j/HCR1 Cooperates with its Structurally Conserved Binding Partner eIF3b/PRT1-RRM and with eIF1A in Stringent AUG Selection

Despite recent progress in our understanding of the numerous functions of individual subunits of eukaryotic translation initiation factor (eIF) 3, little is known on the molecular level. Using NMR spectroscopy, we determined the first solution structure of an interaction between eIF3 subunits. We revealed that a conserved tryptophan residue in the human eIF3j N-terminal acidic motif (NTA) is held in the helix α1 and loop 5 hydrophobic pocket of the human eIF3b RNA recognition motif (RRM). Mutating the corresponding “pocket” residues in its yeast orthologue reduces cellular growth rate, eliminates eIF3j/HCR1 association with eIF3b/PRT1 in vitro and in vivo, affects 40S occupancy of eIF3, and produces a leaky scanning defect indicative of a deregulation of the AUG selection process. Unexpectedly, we found that the N-terminal half of eIF3j/HCR1 containing the NTA is indispensable and sufficient for wild-type growth of yeast cells. Furthermore, we demonstrate that deletion of either j/HCR1 or its N-terminal half only, or mutation of the key tryptophan residues results in the severe leaky scanning phenotype partially suppressible by overexpressed eIF1A, which is thought to stabilize properly formed preinitiation complexes at the correct start codon. These findings indicate that eIF3j/HCR1 remains associated with the scanning preinitiation complexes and does not dissociate from the small ribosomal subunit upon mRNA recruitment, as previously believed. Finally, we provide further support for earlier mapping of the ribosomal binding site for human eIF3j by identifying specific interactions of eIF3j/HCR1 with small ribosomal proteins RPS2 and RPS23 located in the vicinity of the mRNA entry channel. Taken together, we propose that eIF3j/HCR1 closely cooperates with the eIF3b/PRT1 RRM and eIF1A on the ribosome to ensure proper formation of the scanning-arrested conformation required for stringent AUG recognition.     

Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast

Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo.     

Eukaryotic initiation factor (eIF) 1 carries two distinct eIF5-binding faces important for multifactor assembly and AUG selection

Eukaryotic initiation factor (eIF) 1 is a small protein (12 kDa) governing fidelity in translation initiation. It is recruited to the 40 S subunit in a multifactor complex with Met-tRNA(i)(Met), eIF2, eIF3, and eIF5 and binds near the P-site. eIF1 release in response to start codon recognition is an important signal to produce an 80 S initiation complex. Although the ribosome-binding face of eIF1 was identified, interfaces to other preinitiation complex components and their relevance to eIF1 function have not been determined. Exploiting the solution structure of yeast eIF1, here we locate the binding site for eIF5 in its N-terminal tail and at a basic/hydrophobic surface area termed KH, distinct from the ribosome-binding face. Genetic and biochemical studies indicate that the eIF1 N-terminal tail plays a stimulatory role in cooperative multifactor assembly. A mutation altering the basic part of eIF1-KH is lethal and shows a dominant phenotype indicating relaxed start codon selection. Cheung et al. recently demonstrated that the alteration of hydrophobic residues of eIF1 disrupts a critical link to the preinitiation complex that suppresses eIF1 release before start codon selection (Cheung, Y.-N., Maag, D., Mitchell, S. F., Fekete, C. A., Algire, M. A., Takacs, J. E., Shirokikh, N., Pestova, T., Lorsch, J. R., and Hinnebusch, A. (2007) Genes Dev. 21, 1217-1230 ). Interestingly, eIF1-KH includes the altered hydrophobic residues. Thus, eIF5 is an excellent candidate for the direct partner of eIF1-KH that mediates the critical link. The direct interaction at eIF1-KH also places eIF5 near the decoding site of the 40 S subunit.     

Communication between eukaryotic translation initiation factors 5 and 1A within the ribosomal pre-initiation complex plays a role in start site selection

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex scans the mRNA in search of an AUG codon at which to begin translation. Start codon recognition halts scanning and triggers a number of events that commit the complex to beginning translation at that point on the mRNA. Previous studies in vitro and in vivo have indicated that eukaryotic initiation factors (eIFs) 1, 2 and 5 play key roles in these events. In addition, it was reported recently that the C-terminal domain of eIF1A is involved in maintaining the fidelity of start codon recognition. The molecular mechanisms by which these factors work together to ensure fidelity of start site selection remain poorly understood. Here, we report the quantitative characterization of energetic interactions between eIF1A, eIF5 and the AUG codon in an in vitro reconstituted yeast translation initiation system. Our results show that recognition of an AUG codon by the 43 S complex triggers an interaction between eIF5 and eIF1A, resulting in a shift in the equilibrium between two states of the pre-initiation complex. This AUG-dependent change may be a reorganization from a scanning-competent state to a scanning-incompetent state. Mutations in both eIF1A and eIF5 that increase initiation at non-AUG codons in vivo weaken the interaction between the two factors upon AUG recognition, while specifically strengthening it in response to a UUG codon. These data suggest strongly that the interaction between eIF1A and eIF5 is involved in maintaining the fidelity of start codon recognition in vivo.     

Visibly stressed: the role of eIF2, TIA-1, and stress granules in protein translation

Eukaryotic cells express a family of eukaryotic translation initiation factor 2 alpha (eIF2alpha) kinases (eg, PKR, PERK-PEK, GCN2, HRI) that are individually activated in response to distinct types of environmental stress. Phosphorylation of eIF2alpha by one or more of these kinases reduces the concentration of eIF2-guanosine triphosphate (GTP)-transfer ribonucleic acid for methionine (tRNA(Met)), the ternary complex that loads tRNA(Met) onto the small ribosomal subunit to initiate protein translation. When ternary complex levels are reduced, the related RNA-binding proteins TIA-1 and TIAR promote the assembly of a noncanonical preinitiation complex that lacks eIF2-GTP-tRNA(Met). The TIA proteins dynamically sort these translationally incompetent preinitiation complexes into discrete cytoplasmic domains known as stress granules (SGs). RNA-binding proteins that stabilize or destabilize messenger RNA (mRNA) are also recruited to SGs during stress. Thus, TIA-1 and TIAR act downstream of eIF2alpha phosphorylation to promote SG assembly and facilitate mRNA triage during stress. The role of the SG in the integration of translational efficiency, mRNA stability, and the stress response is discussed.     

Large-scale movement of eIF3 domains during translation initiation modulate start codon selection

The eukaryotic initiation factor 3 (eIF3) complex is involved in every step of translation initiation, but there is limited understanding of its molecular functions. Here, we present a single particle electron cryomicroscopy (cryo-EM) reconstruction of yeast 48S ribosomal preinitiation complex (PIC) in an open conformation conducive to scanning, with core subunit eIF3b bound on the 40S interface near the decoding center in contact with the ternary complex eIF2·GTP·initiator tRNA. eIF3b is relocated together with eIF3i from their solvent interface locations observed in other PIC structures, with eIF3i lacking 40S contacts. Re-processing of micrographs of our previous 48S PIC in a closed state also suggests relocation of the entire eIF3b-3i-3g-3a-Cter module during the course of initiation. Genetic analysis indicates that high fidelity initiation depends on eIF3b interactions at the 40S subunit interface that promote the closed PIC conformation, or facilitate the relocation of eIF3b/eIF3i to the solvent interface, on start codon selection.     

Free initiation factors eIF4A and eIF4B are dispensable for translation initiation on uncapped mRNAs

The formation of ribosomal 48S initiation complexes at the start AUG codon of uncapped mRNA leader sequences was studied using the methodology of primer extension inhibition (toe-printing). The experiments were performed in the system composed of purified individual components required for translation initiation. The formation of ribosomal 48S initiation complexes at the initiation codon was tested depending on the presence of the initiation factors eIF4F, eIF4A, and eIF4B. Several mRNAs containing short leader sequences lacking the extended secondary structure were studied. It was found that 48S ribosomal complexes at mRNAs with such leaders were not formed in the absence of eIF4F. In contrast, the removal of either eIF4A or eIF4B from the experimental system was found to be dispensable for the formation of the 48S complex.     

The yeast eukaryotic initiation factor 4G (eIF4G) HEAT domain interacts with eIF1 and eIF5 and is involved in stringent AUG selection

Eukaryotic initiation factor 4G (eIF4G) promotes mRNA recruitment to the ribosome by binding to the mRNA cap- and poly(A) tail-binding proteins eIF4E and Pap1p. eIF4G also binds eIF4A at a distinct HEAT domain composed of five stacks of antiparallel alpha-helices. The role of eIF4G in the later steps of initiation, such as scanning and AUG recognition, has not been defined. Here we show that the entire HEAT domain and flanking residues of Saccharomyces cerevisiae eIF4G2 are required for the optimal interaction with the AUG recognition factors eIF5 and eIF1. eIF1 binds simultaneously to eIF4G and eIF3c in vitro, as shown previously for the C-terminal domain of eIF5. In vivo, co-overexpression of eIF1 or eIF5 reverses the genetic suppression of an eIF4G HEAT domain Ts(-) mutation by eIF4A overexpression. In addition, excess eIF1 inhibits growth of a second eIF4G mutant defective in eIF4E binding, which was also reversed by co-overexpression of eIF4A. Interestingly, excess eIF1 carrying the sui1-1 mutation, known to relax the accuracy of start site selection, did not inhibit the growth of the eIF4G mutant, and sui1-1 reduced the interaction between eIF4G and eIF1 in vitro. Moreover, a HEAT domain mutation altering eIF4G moderately enhances translation from a non-AUG codon. These results strongly suggest that the binding of the eIF4G HEAT domain to eIF1 and eIF5 is important for maintaining the integrity of the scanning ribosomal preinitiation complex.