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.     

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.     

Pi release from eIF2, not GTP hydrolysis, is the step controlled by start-site selection during eukaryotic translation initiation

Irreversible GTP hydrolysis by eIF2 is a critical step in translation initiation in eukaryotes because it is thought to commit the translational machinery to assembling the ribosomal complex at the selected point in the mRNA. Our quantitative analysis of the steps and interactions involved in activating GTP hydrolysis by eIF2 during translation initiation in vitro indicates that a structural rearrangement in the 43S preinitiation complex activates it to become fully competent to hydrolyze GTP. Contrary to the prevailing model, release of inorganic phosphate after GTP hydrolysis by eIF2, not hydrolysis itself, is controlled by recognition of the AUG codon. Release of P(i), which makes GTP hydrolysis irreversible, appears to be controlled by the AUG-dependent dissociation of eIF1 from the preinitiation complex.     

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.     

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.     

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.     

Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex

eIF5 is the GTPase activating protein (GAP) for the eIF2·GTP·Met-tRNA_i^Met ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which P_i is released from eIF2·GDP·P_i. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and P_i release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of P_i release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui⁻ mutations in numerous factors. We conclude that both of eIF5''s functions, regulating P_i release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.     

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.     

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.     

Uncoupling of initiation factor eIF5B/IF2 GTPase and translational activities by mutations that lower ribosome affinity

Translation initiation factor eIF5B/IF2 is a GTPase that promotes ribosomal subunit joining. We show that eIF5B mutations in Switch I, an element conserved in all GTP binding domains, impair GTP hydrolysis and general translation but not eIF5B subunit joining function. Intragenic suppressors of the Switch I mutation restore general translation, but not eIF5B GTPase activity. These suppressor mutations reduce the ribosome affinity of eIF5B and increase AUG skipping/leaky scanning. The uncoupling of translation and eIF5B GTPase activity suggests a regulatory rather than mechanical function for eIF5B GTP hydrolysis in translation initiation. The translational defect suggests eIF5B stabilizes Met-tRNA(i)(Met) binding and that GTP hydrolysis by eIF5B is a checkpoint monitoring 80S ribosome assembly in the final step of translation initiation.     

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.     

Archaeal translation initiation factor aIF2 can substitute for eukaryotic eIF2 in ribosomal scanning during mammalian 48S complex formation

Heterotrimeric translation initiation factor (IF) a/eIF2 (archaeal/eukaryotic IF 2) is present in both Eukarya and Archaea. Despite strong structural similarity between a/eIF2 orthologs from the two domains of life, their functional relationship is obscure. Here, we show that aIF2 from Sulfolobus solfataricus can substitute for its mammalian counterpart in the reconstitution of eukaryotic 48S initiation complexes from purified components. aIF2 is able to correctly place the initiator Met-tRNA_i into the P-site of the 40S ribosomal subunit and accompany the entire set of eukaryotic translation IFs in the process of cap-dependent scanning and AUG codon selection. However, it seems to be unable to participate in the following step of ribosomal subunit joining. In accordance with this, aIF2 inhibits rather than stimulates protein synthesis in mammalian cell-free system. The ability of recombinant aIF2 protein to direct ribosomal scanning suggests that some archaeal mRNAs may utilize this mechanism during translation initiation.     

Translation Initiation Factor eIF4B Interacts with a Picornavirus Internal Ribosome Entry Site in both 48S and 80S Initiation Complexes Independently of Initiator AUG Location

Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation.     

Isolation and functional characterization of a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae in translation initiation factor eIF5: an eIF5-dependent cell-free translation system

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S ribosomal initiation complex (40S.eIF3.AUG.Met-tRNA(f).eIF2.GTP) to promote the hydrolysis of bound GTP. In Saccharomyces cerevisiae, eIF5, a protein of 45346Da, is encoded by a single-copy essential gene, TIF5. In this paper, we have isolated a temperature-sensitive S. cerevisiae strain, TMY5-1, by replacing the wild-type chromosomal copy of TIF5 with one mutagenized in vitro. The mutant yeast cells rapidly cease protein synthesis when grown under non-permissive conditions, lose polyribosomes and accumulate free 80S ribosomes. Further characterization of mutant eIF5 showed that the mutant protein, expressed in Escherichia coli, is defective both in its interaction with eIF2 as well as in mediating the hydrolysis of GTP bound to the 40S initiation complex and consequently in the formation of the 80S initiation complex. Additionally, the availability of a yeast strain containing temperature-sensitive mutation in the eIF5 gene allowed us to construct a cell-free translation system that was dependent on exogenously added eIF5 for translation of mRNAs in vitro.     

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.     

Conformational changes in the P site and mRNA entry channel evoked by AUG recognition in yeast translation preinitiation complexes

The translation preinitiation complex (PIC) is thought to assume an open conformation when scanning the mRNA leader, with AUG recognition evoking a closed conformation and more stable P site interaction of Met-tRNA_i; however, physical evidence is lacking that AUG recognition constrains interaction of mRNA with the 40S binding cleft. We compared patterns of hydroxyl radical cleavage of rRNA by Fe(II)-BABE tethered to unique sites in eIF1A in yeast PICs reconstituted with mRNA harboring an AUG or near-cognate (AUC) start codon. rRNA residues in the P site display reduced cleavage in AUG versus AUC PICs; and enhanced cleavage in the AUC complexes was diminished by mutations of scanning enhancer elements of eIF1A that increase near-cognate recognition in vivo. This suggests that accessibility of these rRNA residues is reduced by accommodation of Met-tRNA_i in the P site (P_IN state) and by their interactions with the anticodon stem of Met-tRNA_i. Our cleavage data also provide evidence that AUG recognition evokes dissociation of eIF1 from its 40S binding site, ejection of the eIF1A-CTT from the P-site and rearrangement to a closed conformation of the entry channel with reduced mobility of mRNA.     

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.     

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.     

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.     

Molecular Mechanism of Scanning and Start Codon Selection in Eukaryotes

Summary: The correct translation of mRNA depends critically on the ability to initiate at the right AUG codon. For most mRNAs in eukaryotic cells, this is accomplished by the scanning mechanism, wherein the small (40S) ribosomal subunit attaches to the 5′ end of the mRNA and then inspects the leader base by base for an AUG in a suitable context, using complementarity with the anticodon of methionyl initiator tRNA (Met-tRNA_i^Met) as the key means of identifying AUG. Over the past decade, a combination of yeast genetics, biochemical analysis in reconstituted systems, and structural biology has enabled great progress in deciphering the mechanism of ribosomal scanning. A robust molecular model now exists, describing the roles of initiation factors, notably eukaryotic initiation factor 1 (eIF1) and eIF1A, in stabilizing an “open” conformation of the 40S subunit with Met-tRNA_i^Met bound in a low-affinity state conducive to scanning and in triggering rearrangement into a “closed” conformation incompatible with scanning, which features Met-tRNA_i^Met more tightly bound to the “P” site and base paired with AUG. It has also emerged that multiple DEAD-box RNA helicases participate in producing a single-stranded “landing pad” for the 40S subunit and in removing the secondary structure to enable the mRNA to traverse the 40S mRNA-binding channel in the single-stranded form for base-by-base inspection in the P site.