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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

rRNA Suppressor of a Eukaryotic Translation Initiation Factor 5B/Initiation Factor 2 Mutant Reveals a Binding Site for Translational GTPases on the Small Ribosomal Subunit

The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.     

The C-Terminal Region of Eukaryotic Translation Initiation Factor 3a (eIF3a) Promotes mRNA Recruitment,Scanning, and,Together with eIF3j and the eIF3b RNA Recognition Motif,Selection of AUG Start Codons

The C-terminal domain (CTD) of the a/Tif32 subunit of budding yeast eukaryotic translation initiation factor 3 (eIF3) interacts with eIF3 subunits j/Hcr1 and b/Prt1 and can bind helices 16 to 18 of 18S rRNA, suggesting proximity to the mRNA entry channel of the 40S subunit. We have identified substitutions in the conserved Lys-Glu-Arg-Arg (KERR) motif and in residues of the nearby box6 element of the a/Tif32 CTD that impair mRNA recruitment by 43S preinitiation complexes (PICs) and confer phenotypes indicating defects in scanning and start codon recognition. The normally dispensable CTD of j/Hcr1 is required for its binding to a/Tif32 and to mitigate the growth defects of these a/Tif32 mutants, indicating physical and functional interactions between these two domains. The a/Tif32 CTD and the j/Hcr1 N-terminal domain (NTD) also interact with the RNA recognition motif (RRM) in b/Prt1, and mutations in both subunits that disrupt their interactions with the RRM increase leaky scanning of an AUG codon. These results, and our demonstration that the extreme CTD of a/Tif32 binds to Rps2 and Rps3, lead us to propose that the a/Tif32 CTD directly stabilizes 43S subunit-mRNA interaction and that the b/Prt1-RRM-j/Hcr1-a/Tif32-CTD module binds near the mRNA entry channel and regulates the transition between scanning-conducive and initiation-competent conformations of the PIC.Eukaryotic translation initiation factor 3 (eIF3) is a multisubunit protein complex that has been implicated in several steps of the translation initiation pathway (reviewed in reference 19). These steps include recruitment of the eIF2-GTP-Met-ternary complex (TC) and other eIFs to the small (40S) ribosomal subunit to form the 43S preinitiation complex (PIC), mRNA recruitment by the 43S PIC, and subsequent scanning of the 5′ untranslated region (UTR) for an AUG start codon. The eIF3 in the budding yeast Saccharomyces cerevisiae is composed of only 6 subunits (a/Tif32, b/Prt1, c/Nip1, i/Tif34, g/Tif35, and j/Hcr1), which have homologs in the larger, 13-subunit eIF3 complex in mammals. Yeast eIF3 can be purified with the TC, eIF1, and eIF5 in a ribosome-free assembly called the multifactor complex (MFC) (2), whose formation appears to promote assembly or stability of the 43S PIC and to stimulate scanning and AUG selection (10, 23, 32, 42, 48, 49, 51).In mammals, there is evidence that eIF3 enhances recruitment of mRNA by interacting directly with eIF4G, the “scaffold” subunit of mRNA cap-binding complex eIF4F, and forming a protein bridge between mRNA and the 43S PIC (24, 25, 35). In budding yeast, direct eIF3-eIF4G interaction has not been detected, and the eIF3-binding domain (25) is not evident in yeast eIF4G. Moreover, depletion of eIF3, but not eIF4G, from yeast cells provokes a strong decrease in the amount of an mRNA (RPL41A) associated with native PICs (23). However, since depletion of eIF3 also reduced the amounts of other MFC components associated with PICs, it remained unclear whether eIF3 acts directly in mRNA recruitment.In favor of a direct role for eIF3, cross-linking analysis of reconstituted mammalian 48S PICs identified contacts of subunits eIF3a and eIF3d with mRNA residues 8 to 17 nucleotides (nt) upstream of the AUG codon, suggesting that these subunits form an extension of the mRNA exit channel (37). Consistent with this, we found that the N-terminal domain (NTD) of yeast a/Tif32 binds Rps0A, located near the mRNA exit pore, and functionally interacts with sequences 5′ to the regulatory upstream open reading frame 1 (uORF1) in GCN4 mRNA (42). Despite these advances, in vivo evidence supporting a direct role of eIF3 in mRNA recruitment by 43S PICs is lacking.Recently, there has been progress in elucidating the molecular mechanisms involved in ribosomal scanning and AUG selection. Reconstituted mammalian 43S PICs containing only eIF1, -1A, and -3 and the TC can scan the leader of an unstructured message and form a stable 48S PIC at the 5′-proximal AUG codon (35). eIF1 and -1A are thought to promote scanning by stabilizing an open conformation of the 40S subunit (6, 13, 26, 27), which appears to involve opening the “latch” on the mRNA entry channel formed by helices 18 and 34 of 18S rRNA (33). eIF1A also promotes a mode of TC binding conducive to scanning (39) and seems to prevent full accommodation of Met-in the P site at non-AUG codons (53). The GTP bound to eIF2 is hydrolyzed, in a manner stimulated by eIF5, but release of phosphate (P_i) from eIF2-GDP-P_i is blocked by eIF1 (1). Entry of AUG into the P site triggers relocation of eIF1 from its binding site on the 40S subunit (27), allowing P_i release (1) and stabilizing the closed, scanning-arrested conformation of the 40S subunit (33).Mutations in eIF1 and eIF1A that reduce the stringency of start codon recognition have been isolated by their ability to increase initiation at a UUG codon in his4 alleles lacking the AUG start codon (the Sui⁻ phenotype) (6, 12, 13, 29, 38, 39, 52). eIF1A mutations with the opposite effect of lowering UUG initiation in the presence of a different Sui⁻ mutation (the Ssu⁻ phenotype) were also obtained (13, 39). Previously, we identified Sui⁻ and Ssu⁻ mutations in the N-terminal domain of eIF3 subunit c/Nip1, which alter its contacts with eIF1, -2, and -5, suggesting that integrity of the MFC is important for the accuracy of AUG selection (49).Several genetic findings also implicate eIF3 in the efficiency of scanning and AUG recognition. The prt1-1 point mutation in b/Prt1 (S518F) (11) impairs translational control of GCN4 mRNA in a manner suggesting a reduced rate of scanning between the short uORFs involved in this control mechanism (30). Disrupting an interaction between a hydrophobic pocket of the noncanonical RNA recognition motif (RRM) in the N terminus of b/Prt1 (henceforth referred to as b/RRM) and a Trp residue in the N-terminal acidic motif of j/Hcr1 (Trp-37) severely reduces the efficiency of initiation at the AUG of uORF1 in GCN4 mRNA, the phenomenon of leaky scanning, implicating the connection between the b/RRM and j/Hcr1 NTD (henceforth referred to as j/NTD) in efficient AUG recognition (10). Similarly, a multiple Ala substitution in RNP1 of the b/RRM evoked leaky scanning of the AUG codon of GCN4 uORF1 (uAUG-1) (32).Interestingly, besides the b/RRM-j/NTD contact, the b/RRM can simultaneously bind to the j/Hcr1-like domain (HLD) in a/Tif32, and j/Hcr1 also independently binds a/Tif32 (50). This network of interactions involving the b/RRM, a/Tif32-HLD, and j/Hcr1 segments was shown to stabilize an eIF3 subassembly (50), referred to below as the b/RRM-j/Hcr1-a/Tif32-CTD module; however, it was not known whether the a/Tif32 HLD component of this module also participates in AUG recognition or other specific steps of initiation.In this report, we provide evidence that the evolutionarily conserved KERR motif in the a/Tif32 HLD (hereafter referred to as a/HLD) functions to enhance mRNA recruitment by 43S PICs, processivity of scanning, and the efficiency of AUG recognition. The identification of Ssu⁻ phenotypes for both KERR mutations and replacement of a nearby element (box6) further implicates the a/HLD in promoting the closed, scanning-arrested conformation of the PIC at start codons. Combining these results with our finding that the a/Tif32 CTD binds the 40S proteins Rps3 and Rps2 and the recent evidence that j/Hcr1 promotes AUG recognition and binds Rps2 leads us to propose that the a/HLD is positioned near the 40S mRNA entry channel, where it promotes mRNA binding and, together with j/Hcr1 and the b/RRM, modulates the transition between the open and closed conformations of the PIC during scanning and AUG recognition.     

The eIF1A solution structure reveals a large RNA-binding surface important for scanning function

The translation initiation factor eIF1A is necessary for directing the 43S preinitiation complex from the 5' end of the mRNA to the initiation codon in a process termed scanning. We have determined the solution structure of human eIF1A, which reveals an oligonucleotide-binding (OB) fold and an additional domain. NMR titration experiments showed that eIF1A binds single-stranded RNA oligonucleotides in a site-specific, but non-sequence-specific manner, hinting at an mRNA interaction rather than specific rRNA or tRNA binding. The RNA binding surface extends over a large area covering the canonical OB fold binding site as well as a groove leading to the second domain. Site-directed mutations at multiple positions along the RNA-binding surface were defective in the ability to properly assemble preinitiation complexes at the AUG codon in vitro.     

uS5/Rps2 residues at the 40S ribosome entry channel enhance initiation at suboptimal start codons in vivo

The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S subunit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that nonlethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo.     

Interaction of the RNP1 motif in PRT1 with HCR1 promotes 40S binding of eukaryotic initiation factor 3 in yeast

We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1 (prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation in vivo and reduces 40S-binding of eIF3 to native preinitiation complexes. Several findings indicate that the rnp1 lesion decreases recruitment of eIF3 to the 40S subunit by HCR1: (i) rnp1 strongly impairs the association of HCR1 with PRT1 without substantially disrupting the eIF3 complex; (ii) rnp1 impairs the 40S binding of eIF3 more so than the 40S binding of HCR1; (iii) overexpressing HCR1-R215I decreases the Ts(-) phenotype and increases 40S-bound eIF3 in rnp1 cells; (iv) the rnp1 Ts(-) phenotype is exacerbated by tif32-Delta6, which eliminates a binding determinant for HCR1 in TIF32; and (v) hcr1Delta impairs 40S binding of eIF3 in otherwise wild-type cells. Interestingly, rnp1 also reduces the levels of 40S-bound eIF5 and eIF1 and increases leaky scanning at the GCN4 uORF1. Thus, the PRT1 RNP1 motif coordinates the functions of HCR1 and TIF32 in 40S binding of eIF3 and is needed for optimal preinitiation complex assembly and AUG 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.     

N- and C-terminal residues of eIF1A have opposing effects on the fidelity of start codon selection

Translation initiation factor eIF1A stimulates preinitiation complex (PIC) assembly and scanning, but the molecular mechanisms of its functions are not understood. We show that the F131A,F133A mutation in the C-terminal tail (CTT) of eIF1A impairs recruitment of the eIF2-GTP-Met-tRNA(i)(Met) ternary complex to 40S subunits, eliminating functional coupling with eIF1. Mutating residues 17-21 in the N-terminal tail (NTT) of eIF1A also reduces PIC assembly, but in a manner rescued by eIF1. Interestingly, the 131,133 CTT mutation enhances initiation at UUG codons (Sui(-) phenotype) and decreases leaky scanning at AUG, while the NTT mutation 17-21 suppresses the Sui(-) phenotypes of eIF5 and eIF2beta mutations and increases leaky scanning. These findings and the opposite effects of the mutations on eIF1A binding to reconstituted PICs suggest that the NTT mutations promote an open, scanning-conducive conformation of the PIC, whereas the CTT mutations 131,133 have the reverse effect. We conclude that tight binding of eIF1A to the PIC is an important determinant of AUG selection and is modulated in opposite directions by residues in the NTT and CTT of eIF1A.     

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.     

Rps5-Rps16 communication is essential for efficient translation initiation in yeast S. cerevisiae

Conserved ribosomal proteins frequently harbor additional segments in eukaryotes not found in bacteria, which could facilitate eukaryotic-specific reactions in the initiation phase of protein synthesis. Here we provide evidence showing that truncation of the N-terminal domain (NTD) of yeast Rps5 (absent in bacterial ortholog S7) impairs translation initiation, cell growth and induction of GCN4 mRNA translation in a manner suggesting incomplete assembly of 48S preinitiation complexes (PICs) at upstream AUG codons in GCN4 mRNA. Rps5 mutations evoke accumulation of factors on native 40S subunits normally released on conversion of 48S PICs to 80S initiation complexes (ICs) and this abnormality and related phenotypes are mitigated by the SUI5 variant of eIF5. Remarkably, similar effects are observed by substitution of Lys45 in the Rps5-NTD, involved in contact with Rps16, and by eliminating the last two residues of the C-terminal tail (CTT) of Rps16, believed to contact initiator tRNA base-paired to AUG in the P site. We propose that Rps5-NTD-Rps16-NTD interaction modulates Rps16-CTT association with Met-tRNA_i^Met to promote a functional 48S PIC.