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.     

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.     

Identification of compounds that decrease the fidelity of start codon recognition by the eukaryotic translational machinery

Translation initiation in eukaryotes involves more than a dozen protein factors. Alterations in six factors have been found to reduce the fidelity of start codon recognition by the ribosomal preinitiation complex in yeast, a phenotype referred to as Sui(-). No small molecules are known that affect the fidelity of start codon recognition. Such compounds would be useful tools for probing the molecular mechanics of translation initiation and its regulation. To find compounds with this effect, we set up a high-throughput screen using a dual luciferase assay in S. cerevisiae. Screening of over 55,000 compounds revealed two structurally related molecules that decrease the fidelity of start codon selection by approximately twofold in the dual luciferase assay. This effect was confirmed using additional in vivo assays that monitor translation from non-AUG start codons. Both compounds increase translation of a natural upstream open reading frame previously shown to initiate translation at a UUG. The compounds were also found to exacerbate increased use of UUG as a start codon (Sui(-) phenotype) conferred by haploinsufficiency of wild-type eukaryotic initiation factor (eIF) 1, or by mutation in eIF1. Furthermore, the effects of the compounds are suppressed by overexpressing eIF1, which is known to restore the fidelity of start codon selection in strains harboring Sui(-) mutations in various other initiation factors. Together, these data strongly suggest that the compounds affect the translational machinery itself to reduce the accuracy of selecting AUG as the start codon.     

eIF1 Controls Multiple Steps in Start Codon Recognition during Eukaryotic Translation Initiation

Eukaryotic translation initiation factor (eIF) 1 is a central mediator of start codon recognition. Dissociation of eIF1 from the preinitiation complex (PIC) allows release of phosphate from the G-protein factor eIF2, triggering downstream events in initiation. Mutations that weaken binding of eIF1 to the PIC decrease the fidelity of start codon recognition (Sui⁻ phenotype) by allowing increased eIF1 release at non-AUG codons. Consistent with this, overexpression of these mutant proteins suppresses their Sui⁻ phenotypes. Here, we have examined mutations at the penultimate residue of eIF1, G107, that produce Sui⁻ phenotypes without increasing the rate of eIF1 release. We provide evidence that, in addition to its role in gating phosphate release, dissociation of eIF1 triggers conversion from an open, scanning-competent state of the PIC to a stable, closed one. We also show that eIF5 antagonizes binding of eIF1 to the complex and that key interactions of eIF1 with its partners are modulated by the charge at and around G107. Our data indicate that eIF1 plays multiple roles in start codon recognition and suggest that prior to AUG recognition it prevents eIF5 from binding to a key site in the PIC required for triggering downstream events.     

Functional Characterization of the Role of the N-terminal Domain of the c/Nip1 Subunit of Eukaryotic Initiation Factor 3 (eIF3) in AUG Recognition

In eukaryotes, for a protein to be synthesized, the 40 S subunit has to first scan the 5'-UTR of the mRNA until it has encountered the AUG start codon. Several initiation factors that ensure high fidelity of AUG recognition were identified previously, including eIF1A, eIF1, eIF2, and eIF5. In addition, eIF3 was proposed to coordinate their functions in this process as well as to promote their initial binding to 40 S subunits. Here we subjected several previously identified segments of the N-terminal domain (NTD) of the eIF3c/Nip1 subunit, which mediates eIF3 binding to eIF1 and eIF5, to semirandom mutagenesis to investigate the molecular mechanism of eIF3 involvement in these reactions. Three major classes of mutant substitutions or internal deletions were isolated that affect either the assembly of preinitiation complexes (PICs), scanning for AUG, or both. We show that eIF5 binds to the extreme c/Nip1-NTD (residues 1-45) and that impairing this interaction predominantly affects the PIC formation. eIF1 interacts with the region (60-137) that immediately follows, and altering this contact deregulates AUG recognition. Together, our data indicate that binding of eIF1 to the c/Nip1-NTD is equally important for its initial recruitment to PICs and for its proper functioning in selecting the translational start site.     

Regulation of GTP hydrolysis prior to ribosomal AUG selection during eukaryotic translation initiation

Genetic studies in yeast have shown that the translation initiation factor eIF5 plays an important role in the selection of the AUG start codon. In order to ensure translation fidelity, the hydrolysis of GTP bound to the 40S preinitiation complex (40S.Met-tRNA(i).eIF2.GTP), promoted by eIF5, must occur only when the complex has selected the AUG start codon. However, the mechanism that prevents the eIF5-promoted GTP hydrolysis, prior to AUG selection by the ribosomal machinery, is not known. In this work, we show that the presence of initiation factors eIF1, eIF1A and eIF3 in the 40S preinitiation complex (40S.eIF1.eIF1A.eIF3.Met-tRNA(i).eIF2.GTP) and the subsequent binding of the preinitiation complex to eIF4F bound at the 5'-cap structure of mRNA are necessary for preventing eIF5-promoted hydrolysis of GTP in the 40S preinitiation complex. This block in GTP hydrolysis is released upon AUG selection by the 40S preinitiation complex. These results, taken together, demonstrate the biochemical requirements for regulation of GTP hydrolysis and its coupling to the AUG selection process during translation initiation.     

Sliding of a 43S ribosomal complex from the recognized AUG codon triggered by a delay in eIF2-bound GTP hydrolysis

During eukaryotic translation initiation, 43S ribosomal complex scans mRNA leader unless an AUG codon in an appropriate context is found. Establishing the stable codon–anticodon base-pairing traps the ribosome on the initiator codon and triggers structural rearrangements, which lead to P_i release from the eIF2-bound GTP. It is generally accepted that AUG recognition by the scanning 43S complex sets the final point in the process of start codon selection, while latter stages do not contribute to this process. Here we use translation reconstitution approach and kinetic toe-printing assay to show that after the 48S complex is formed on an AUG codon, in case GTP hydrolysis is impaired, the ribosomal subunit is capable to resume scanning and slides downstream to the next AUG. In contrast to leaky scanning, this sliding is not limited to AUGs in poor nucleotide contexts and occurs after a relatively long pause at the recognized AUG. Thus, recognition of an AUG per se does not inevitably lead to this codon being selected for initiation of protein synthesis. Instead, it is eIF5-induced GTP hydrolysis and P_i release that irreversibly trap the 48S complex, and this complex is further stabilized by eIF5B and 60S joining.     

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.     

Specific domains in yeast translation initiation factor eIF4G strongly bias RNA unwinding activity of the eIF4F complex toward duplexes with 5'-overhangs

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex is recruited to the 5'-end of an mRNA through its interaction with the 7-methylguanosine cap, and it subsequently scans along the mRNA to locate the start codon. Both mRNA recruitment and scanning require the removal of secondary structure within the mRNA. Eukaryotic translation initiation factor 4A is an essential component of the translational machinery thought to participate in the clearing of secondary structural elements in the 5'-untranslated regions of mRNAs. eIF4A is part of the 5'-7-methylguanosine cap-binding complex, eIF4F, along with eIF4E, the cap-binding protein, and the scaffolding protein eIF4G. Here, we show that Saccharomyces cerevisiae eIF4F has a strong preference for unwinding an RNA duplex with a single-stranded 5'-overhang versus the same duplex with a 3'-overhang or without an overhang. In contrast, eIF4A on its own has little RNA substrate specificity. Using a series of deletion constructs of eIF4G, we demonstrate that its three previously elucidated RNA binding domains work together to provide eIF4F with its 5'-end specificity, both by promoting unwinding of substrates with 5'-overhangs and inhibiting unwinding of substrates with 3'-overhangs. Our data suggest that the RNA binding domains of eIF4G provide the S. cerevisiae eIF4F complex with a second mechanism, in addition to the eIF4E-cap interaction, for directing the binding of pre-initiation complexes to the 5'-ends of mRNAs and for biasing scanning in the 5' to 3' direction.     

Crystal structure of the C-terminal domain of S.cerevisiae eIF5

eIF5, a GTPase-activating protein (GAP) specific for eIF2, plays a critical role in pre-initiation complex assembly and correct AUG selection during eukaryotic translation initiation. eIF5 is involved in the formation of the multifactor complex (MFC), an important intermediate of the 43S pre-initiation complex. The C-terminal domain (CTD) of eIF5 functions as the structural core in the MFC assembly. Here we report the 1.5A crystal structure of eIF5-CTD, confirming that eIF5-CTD contains an atypical HEAT motif. In addition, analyzing the electrostatic potential and the distribution of conserved residues on the protein surface, we confirm and suggest some potential regions of interactions between eIF5-CTD and other eIFs. The structure of eIF5-CTD provides useful information in understanding the mechanism of the MFC assembly.     

Stringency of start codon selection modulates autoregulation of translation initiation factor eIF5

An AUG in an optimal nucleotide context is the preferred translation initiation site in eukaryotic cells. Interactions among translation initiation factors, including eIF1 and eIF5, govern start codon selection. Experiments described here showed that high intracellular eIF5 levels reduced the stringency of start codon selection in human cells. In contrast, high intracellular eIF1 levels increased stringency. High levels of eIF5 induced translation of inhibitory upstream open reading frames (uORFs) in eIF5 mRNA that initiate with AUG codons in conserved poor contexts. This resulted in reduced translation from the downstream eIF5 start codon, indicating that eIF5 autoregulates its own synthesis. As with eIF1, which is also autoregulated through translation initiation, features contributing to eIF5 autoregulation show deep evolutionary conservation. The results obtained provide the basis for a model in which auto- and cross-regulation of eIF5 and eIF1 translation establish a regulatory feedback loop that would stabilize the stringency of start codon selection.     

Sequential Eukaryotic Translation Initiation Factor 5 (eIF5) Binding to the Charged Disordered Segments of eIF4G and eIF2β Stabilizes the 48S Preinitiation Complex and Promotes Its Shift to the Initiation Mode

During translation initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eukaryotic translation initiation factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2β bind three common partners, eIF5, eIF1, and mRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2β-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner reversed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2β-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2β mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involving the eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2β and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNA(i)(Met):mRNA binding to the P site.     

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.     

Kinetic analysis of late steps of eukaryotic translation initiation

Little is known about the molecular mechanics of the late events of translation initiation in eukaryotes. We present a kinetic dissection of the transition from a preinitiation complex after start codon recognition to the final 80S initiation complex. The resulting framework reveals that eukaryotic initiation factor (eIF)5B actually accelerates the rate of ribosomal subunit joining, and this acceleration is influenced by the conformation of the GTPase active site of the factor mediated by the bound nucleotide. eIF1A accelerates joining through its C-terminal interaction with eIF5B, and eIF1A release from the initiating ribosome, which occurs only after subunit joining, is accelerated by GTP hydrolysis by eIF5B. Following subunit joining, GTP hydrolysis by eIF5B alters the conformation of the final initiation complex and clears a path to promote rapid release of eIF1A. Our data, coupled with previous work, indicate that eIF1A is present on the ribosome throughout the entire initiation process and plays key roles at every stage.     

Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly

Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1^G107R but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.     

Free energy landscape of RNA binding dynamics in start codon recognition by eukaryotic ribosomal pre-initiation complex

Specific interaction between the start codon, 5’-AUG-3’, and the anticodon, 5’-CAU-3’, ensures accurate initiation of translation. Recent studies show that several near-cognate start codons (e.g. GUG and CUG) can play a role in initiating translation in eukaryotes. However, the mechanism allowing initiation through mismatched base-pairs at the ribosomal decoding site is still unclear at an atomic level. In this work, we propose an extended simulation-based method to evaluate free energy profiles, through computing the distance between each base-pair of the triplet interactions involved in recognition of start codons in eukaryotic translation pre-initiation complex. Our method provides not only the free energy penalty for mismatched start codons relative to the AUG start codon, but also the preferred pathways of transitions between bound and unbound states, which has not been described by previous studies. To verify the method, the binding dynamics of cognate (AUG) and near-cognate start codons (CUG and GUG) were simulated. Evaluated free energy profiles agree with experimentally observed changes in initiation frequencies from respective codons. This work proposes for the first time how a G:U mismatch at the first position of codon (GUG)-anticodon base-pairs destabilizes the accommodation in the initiating eukaryotic ribosome and how initiation at a CUG codon is nearly as strong as, or sometimes stronger than, that at a GUG codon. Our method is expected to be applied to study the affinity changes for various mismatched base-pairs.     

The Eukaryotic Initiation Factor (eIF) 4G HEAT Domain Promotes Translation Re-initiation in Yeast Both Dependent on and Independent of eIF4A mRNA Helicase

Translation re-initiation provides the molecular basis for translational control of mammalian ATF4 and yeast GCN4 mediated by short upstream open reading (uORFs) in response to eIF2 phosphorylation. eIF4G is the major adaptor subunit of eIF4F that binds the cap-binding subunit eIF4E and the mRNA helicase eIF4A and is also required for re-initiation in mammals. Here we show that the yeast eIF4G2 mutations altering eIF4E- and eIF4A-binding sites increase re-initiation at GCN4 and impair recognition of the start codons of uORF1 or uORF4 located after uORF1. The increase in re-initiation at GCN4 was partially suppressed by increasing the distance between uORF1 and GCN4, suggesting that the mutations decrease the migration rate of the scanning ribosome in the GCN4 leader. Interestingly, eIF4E overexpression suppressed both the phenotypes caused by the mutation altering eIF4E-binding site. Thus, eIF4F is required for accurate AUG selection and re-initiation also in yeast, and the eIF4G interaction with the mRNA-cap appears to promote eIF4F re-acquisition by the re-initiating 40 S subunit. However, eIF4A overexpression suppressed the impaired AUG recognition but not the increase in re-initiation caused by the mutations altering eIF4A-binding site. These results not only provide evidence that mRNA unwinding by eIF4A stimulates start codon recognition, but also suggest that the eIF4A-binding site on eIF4G made of the HEAT domain stimulates the ribosomal scanning independent of eIF4A. Based on the RNA-binding activities identified within the unstructured segments flanking the eIF4G2 HEAT domain, we discuss the role of the HEAT domain in scanning beyond loading eIF4A onto the pre-initiation complex.     

Where to begin? The mechanism of translation initiation codon selection in eukaryotes

Selecting the codon at which to begin translation is a complicated event in an already complicated process. Many protein initiation factors (eIFs) have been implicated in start site selection, but the mechanistic details of their activities have remained obscure until recently. Biochemical and genetic studies of eIFs 1, 1A, 2 and 5 have suggested that the 43S pre-initiation complex exists in two conformations and that the changing interactions of the factors within the 43S pre-initiation complex in response to encountering an AUG codon regulates these conformations and, ultimately, the selection of the start codon.     

Interaction between 25S rRNA A Loop and Eukaryotic Translation Initiation Factor 5B Promotes Subunit Joining and Ensures Stringent AUG Selection

In yeast, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During the last step of translation initiation, eukaryotic initiation factor 5B (eIF5B) promotes the 60S subunit joining with the 40S initiation complex (IC). Malfunctional 60S subunits produced by misfolding or mutation may disrupt the 40S IC stalling on the start codon, thereby altering the stringency of initiation. Using several point mutations isolated by random mutagenesis, here we studied the role of 25S rRNA in start codon selection. Three mutations changing bases near the ribosome surface had strong effects, allowing the initiating ribosomes to skip both AUG and non-AUG codons: C2879U and U2408C, altering the A loop and P loop, respectively, of the peptidyl transferase center, and G1735A, mapping near a Eukarya-specific bridge to the 40S subunit. Overexpression of eIF5B specifically suppressed the phenotype caused by C2879U, suggesting functional interaction between eIF5B and the A loop. In vitro reconstitution assays showed that C2879U decreased eIF5B-catalyzed 60S subunit joining with a 40S IC. Thus, eIF5B interaction with the peptidyl transferase center A loop increases the accuracy of initiation by stabilizing the overall conformation of the 80S initiation complex. This study provides an insight into the effect of ribosomal mutations on translation profiles in eukaryotes.     

eIF3j facilitates loading of release factors into the ribosome

eIF3j is one of the eukaryotic translation factors originally reported as the labile subunit of the eukaryotic translation initiation factor eIF3. The yeast homolog of this protein, Hcr1, has been implicated in stringent AUG recognition as well as in controlling translation termination and stop codon readthrough. Using a reconstituted mammalian in vitro translation system, we showed that the human protein eIF3j is also important for translation termination. We showed that eIF3j stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the initiation factor eIF3, which also stimulates peptide release, eIF3j activity in translation termination increases. We found that eIF3j interacts with the pre-termination ribosomal complex, and eRF3 destabilises this interaction. In the solution, these proteins bind to each other and to other participants of translation termination, eRF1 and PABP, in the presence of GTP. Using a toe-printing assay, we determined the stage at which eIF3j functions – binding of release factors to the A-site of the ribosome before GTP hydrolysis. Based on these data, we assumed that human eIF3j is involved in the regulation of translation termination by loading release factors into the ribosome.