Interaction between eukaryotic initiation factors 1A and 5B is required for efficient ribosomal subunit joining

Eukaryotic initiation factor 5B (eIF5B) is a GTPase that facilitates joining of the 60 S ribosomal subunit to the 40 S ribosomal subunit during translation initiation. Formation of the resulting 80 S initiation complex triggers eIF5B to hydrolyze its bound GTP, reducing the affinity of the factor for the complex and allowing it to dissociate. Here we present a kinetic analysis of GTP hydrolysis by eIF5B in the context of the translation initiation pathway. Our data indicate that stimulation of GTP hydrolysis by eIF5B requires the completion of early steps in translation initiation, including the eIF1- and eIF1A-dependent delivery of initiator methionyl-tRNA to the 40 S ribosomal subunit and subsequent GTP hydrolysis by eIF2. Full activation of GTP hydrolysis by eIF5B requires the extreme C terminus of eIF1A, which has previously been shown to interact with the C terminus of eIF5B. Disruption of either isoleucine residue in the eIF1A C-terminal sequence DIDDI reduces the rate constant for GTP hydrolysis by approximately 20-fold, whereas changing the aspartic acid residues has no effect. Changing the isoleucines in the C terminus of eIF1A also disrupts the ability of eIF5B to facilitate subunit joining. These data indicate that the interaction of the C terminus of eIF1A with eIF5B promotes ribosomal subunit joining and possibly provides a checkpoint for correct complex formation, allowing full activation of GTP hydrolysis only upon formation of a properly organized 80 S initiation complex.     

Communication between eukaryotic translation initiation factors 1 and 1A on the yeast small ribosomal subunit

We have used expressed protein ligation to site-specifically label eukaryotic translation initiation factors (eIFs) 1 and 1A at their C termini with tetramethyl rhodamine. These fluorescent proteins were used in steady-state anisotropy-based binding experiments to measure the dissociation constants of the factors and the yeast small (40S) ribosomal subunit for the first time. These studies demonstrate that both eIF1 and eIF1A are capable of binding to the 40S subunit in the absence of any other initiation factors or mRNA, arguing against previous suggestions that eIF3 is required for recruitment of eIF1 to the small ribosomal subunit. Strikingly, the data also demonstrate that there is approximately ninefold thermodynamic coupling in the binding of the two factors to the 40S subunit. This indicates that eIF1 and eIF1A communicate with one another when bound to the 40S subunit. Communication between these two factors is likely to be important for coordinating their functions during the initiation process. The data presented here provide a foundation on which to build a quantitative understanding of the network of interactions between these essential factors and the rest of the initiation machinery.     

Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.

The AUG-dependent formation of an 80 S ribosomal initiation complex was studied using purified rabbit reticulocyte initiation factors radiolabeled by reductive methylation. The radiolabeled initiation factors were as biologically active as untreated factors. Reaction mixtures containing a variety of components (AUG, GTP, Met-tRNAf, initiation factors, and 40 S and 60 S ribosomal subunits) were incubated at 30 degrees C and then analyzed on linear sucrose gradients for the formation of ribosomal complexes. The results show that both eukaryotic initiation factor (eIF)-3 and the ternary complex (eIF-2.GTP.Met-tRNAf) bind independently to the 40 S subunit and each of these components enhances the binding of the other. All of the polypeptides of eIF-2 and eIF-3 participate in this binding. Formation of an 80 S ribosomal complex requires eIF-5 and 60 S subunits in a reaction that is stimulated by eIF-4C. Both eIF-2 and eIF-3 are released from the 40 S preinitiation complex during formation of the 80 S initiation complex. Release of eIF-2 and eIF-3 does not occur and 80 S ribosomal complexes are not formed if GTP is replaced by a nonhydrolyzable analog such as guanosine 5'-O3-(1,2-mu-imido)triphosphate. Despite a variety of attempts, it has not yet been possible to demonstrate binding of eIF-4C, eIF-4D, or eIF-5 to either 40 S or 80 S ribosomal complexes.     

Structural insights into eukaryotic ribosomes and the initiation of translation

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Highlights? Overview of recent progress in the structural characterization of eukaryotic ribosomes and initiation complexes. ? Crystal structures, cryo-EM and biochemical data are combined to derive structural models of larger assemblies. ? Homology models of eukaryotic initiation complexes provide a starting point for future experiments.     

A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors

Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5' leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.     

The eukaryotic translation initiation factors eIF1 and eIF1A induce an open conformation of the 40S ribosome

Initiation of translation is the process by which initiator tRNA and the start codon of mRNA are positioned in the ribosomal P site. In eukaryotes, one of the first steps involves the binding of two small factors, eIF1 and eIF1A, to the small (40S) ribosomal subunit. This facilitates tRNA binding, allows scanning of mRNA, and maintains fidelity of start codon recognition. Using cryo-EM, we have obtained 3D reconstructions of 40S bound to both eIF1 and eIF1A, and with each factor alone. These structures reveal that together, eIF1 and eIF1A stabilize a conformational change that opens the mRNA binding channel. Biochemical data reveal that both factors accelerate the rate of ternary complex (eIF2*GTP*Met-tRNA(i)(Met)) binding to 40S but only eIF1A stabilizes this interaction. Our results suggest that eIF1 and eIF1A promote an open, scanning-competent preinitiation complex that closes upon start codon recognition and eIF1 release to stabilize ternary complex binding and clamp down on mRNA.     

Role of eukaryotic initiation factor 5 in the formation of 80 S initiation complexes.

The function of eukaryotic initiation factor 5 (eIF-5) from rabbit reticulocyte lysate has been studied by sucrose gradient preparation of 40 S and 80 S initiation complexes. eIF-5 is required for transfer of initiator tRNA from 40 S preinitiation complexes to puromycin-reactive 80 S complexes. The transfer is dependent upon GTP hydrolysis and is associated with release of eIF-2 and eIF-3 from the 40 S subunit. The GTP-dependent loss of eIF-2 and eIF-3 is catalyzed by eIF-5 in the absence of 60 S subunits or when subunit joining is prevented by edeine, but not when GTP is replaced by GuoPP(NH)P. Unstable 40 S subunit . Met-tRNAf complexes generated by eIF-5 can form puromycin-reactive 80 S complexes when 60 S subunits are added in the absence of added GTP. In addition, kinetic evidence is presented that indicates GTP hydrolysis occurs prior to 80 S complex formation.     

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.     

Binding of inosine-substituted mRNA to reticulocyte ribosomes and eukaryotic initiation factors 4A and 4B requires ATP

We examined the hypothesis that initiation of eukaryotic protein synthesis involves ATP-dependent melting of 5'-cap-proximal secondary structure in mRNA by eukaryotic initiation factors 4A and 4B. In reticulocyte lysate depleted of ribonucleoside triphosphates by pretreatment with hexokinase/glucose, initiation complex formation by native reovirus mRNA showed a strict requirement for ATP. The corresponding mRNA synthesized with ITP in place of GTP to minimize secondary structure also required ATP for binding to 40 S ribosomal subunits in complexes characteristic of initiation. In a partial reaction without ribosomes, purified eukaryotic initiation factors 4A and 4B bound and cross-linked to the capped 5'-end of oxidized mRNA. This interaction was ATP-dependent with inosine-substituted or bromouridine-containing reovirus RNAs as observed previously with native mRNA. The results indicate that if initiation involves ATP-dependent denaturation of mRNA, the effect must occur after initiation factor-mediated attachment of mRNA to the 40 S ribosomal subunit.     

Distributed control for recruitment, scanning and subunit joining steps of translation initiation

Protein synthesis utilizes a large proportion of the available free energy in the eukaryotic cell and must be precisely controlled, yet up to now there has been no systematic rate control analysis of the in vivo process. We now present a novel study of rate control by eukaryotic translation initiation factors (eIFs) using yeast strains in which chromosomal eIF genes have been placed under the control of the tetO7 promoter system. The results reveal that, contrary to previously published reports, control of the initiation pathway is distributed over all of the eIFs, whereby rate control (the magnitude of their respective component control coefficients) follows the order: eIF4G>eIF1A>eIF4E>eIF5B. The apparent rate control effects of eIFs observed in standard cell-free extract experiments, on the other hand, do not accurately reflect the steady state in vivo data. Overall, this work establishes the first quantitative control framework for the study of in vivo eukaryotic translation.     

Characterization of the p33 subunit of eukaryotic translation initiation factor-3 from Saccharomyces cerevisiae

Eukaryotic translation initiation factor-3 (eIF3) is a large multisubunit complex that binds to the 40 S ribosomal subunit and promotes the binding of methionyl-tRNAi and mRNA. The molecular mechanism by which eIF3 exerts these functions is incompletely understood. We report here the cloning and characterization of TIF35, the Saccharomyces cerevisiae gene encoding the p33 subunit of eIF3. p33 is an essential protein of 30,501 Da that is required in vivo for initiation of protein synthesis. Glucose repression of TIF35 expressed from a GAL1 promoter results in depletion of both the p33 and p39 subunits. Expression of histidine-tagged p33 in yeast in combination with Ni2+ affinity chromatography allows the isolation of a complex containing the p135, p110, p90, p39, and p33 subunits of eIF3. The p33 subunit binds both mRNA and rRNA fragments due to an RNA recognition motif near its C terminus. Deletion of the C-terminal 71 amino acid residues causes loss of RNA binding, but expression of the truncated form as the sole source of p33 nevertheless supports the slow growth of yeast. These results indicate that the p33 subunit of eIF3 plays an important role in the initiation phase of protein synthesis and that its RNA-binding domain is required for optimal activity.     

Position of eukaryotic initiation factor eIF5B on the 80S ribosome mapped by directed hydroxyl radical probing

Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that mediates displacement of initiation factors from the 40S ribosomal subunit in 48S initiation complexes and joining of 40S and 60S subunits. Here, we determined eIF5B's position on 80S ribosomes by directed hydroxyl radical cleavage. In the resulting model, eIF5B is located in the intersubunit cleft of the 80S ribosome: domain 1 is positioned near the GTPase activating center of the 60S subunit, domain 2 interacts with the 40S subunit (helices 3, 5 and the base of helix 15 of 18S rRNA and ribosomal protein (rp) rpS23), domain 3 is sandwiched between subunits and directly contacts several ribosomal elements including Helix 95 of 28S rRNA and helix 44 of 18S rRNA, domain 4 is near the peptidyl-transferase center and its helical subdomain contacts rpL10E. The cleavage data also indicate that binding of eIF5B might induce conformational changes in both subunits, with ribosomal segments wrapping around the factor. Some of these changes could also occur upon binding of other translational GTPases, and may contribute to factor recognition.     

Factor-free translation of poly(dT) by 80S ribosomes from Saccharomyces cerevisiae

The conditions for preparation of 80S ribosomes from S. cerevisiae are suggested. The ribosomes can bind Phe-tRNAPhe in poly(U)-or poly(dT)-directed manner and are shown to be able to translate poly(dT) in the absence of elongation factor and GTP. Effects of different antibiotics on the factor-free translation have been studied.     

High-throughput assays probing protein-RNA interactions of eukaryotic translation initiation factors

Protein-RNA interactions are involved in all facets of RNA biology. The identification of small molecules that selectively block such bimolecular interactions could provide insight into previously unexplored steps of gene regulation. Such is the case for regulation of eukaryotic protein synthesis where interactions between messenger RNA (mRNA) and several eukaryotic initiation factors govern the recruitment of 40S ribosomes (and associated factors) to mRNA templates during the initiation phase. We have designed simple fluorescence polarization-based high-throughput screening assays that query the binding of several translation factors to RNA and found that the mixed inhibitor p-chloromercuribenzoate interferes with poly(A) binding protein-RNA interaction.     

eIF5B employs a novel domain release mechanism to catalyze ribosomal subunit joining

eIF5B is a eukaryal translational GTPase that catalyzes ribosomal subunit joining to form elongation‐competent ribosomes. Despite its central role in protein synthesis, the mechanistic details that govern the function of eIF5B or its archaeal and bacterial (IF2) orthologs remained unclear. Here, we present six high‐resolution crystal structures of eIF5B in its apo, GDP‐ and GTP‐bound form that, together with an analysis of the thermodynamics of nucleotide binding, provide a detailed picture of the entire nucleotide cycle performed by eIF5B. Our data show that GTP binding induces significant conformational changes in the two conserved switch regions of the G domain, resulting in the reorganization of the GTPase center. These rearrangements are accompanied by the rotation of domain II relative to the G domain and release of domain III from its stable contacts with switch 2, causing an increased intrinsic flexibility in the free GTP‐bound eIF5B. Based on these data, we propose a novel domain release mechanism for eIF5B/IF2 activation that explains how eIF5B and IF2 fulfill their catalytic role during ribosomal subunit joining.     

Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry.

Translation of picornavirus RNA is initiated after ribosomal binding to an internal ribosomal entry site (IRES) within the 5' untranslated region. We have reconstituted IRES-mediated initiation on encephalomyocarditis virus RNA from purified components and used primer extension analysis to confirm the fidelity of 48S preinitiation complex formation. Eukaryotic initiation factor 2 (eIF2), eIF3, and eIF4F were required for initiation; eIF4B and to a lesser extent the pyrimidine tract-binding protein stimulated this process. We show that eIF4F binds to the IRES in a novel cap-independent manner and suggest that cap- and IRES-dependent initiation mechanisms utilize different modes of interaction with this factor to promote ribosomal attachment to mRNA.     

Isolation and characterization of ribosomes and translation initiation factors from the gram-positive soil bacterium Streptomyces lividans

A primer extension inhibition (toeprint) assay was developed using ribosomes and ribosomal subunits from Streptomyces lividans. This assay allowed the study of ribosome binding to streptomycete leaderless and leadered mRNA. Purified 30S subunits were unable to form a ternary complex on aph leaderless mRNA, whereas 70S ribosomes could form ternary complexes on this mRNA. 30S subunits formed ternary complexes on leadered aph and malE mRNA. The translation initiation factors (IF1, IF2, and IF3) from S. lividans were isolated and included in toeprint and filter binding assays with leadered and leaderless mRNA. Generally, the IFs reduced the toeprint signal on leadered mRNA; however, incubation of IF1 and IF2 with 30S subunits that had been washed under high-salt conditions promoted the formation of a ternary complex on aph leaderless mRNA. Our data suggest that, as reported for Escherichia coli, initiation complexes with leaderless mRNAs might use a novel pathway involving 70S ribosomes or 30S subunits bound by IF1 and IF2 but not IF3. Some mRNA-ribosome-initiator tRNA reactions that yielded weak or no toeprint signals still formed complexes in filter binding assays, suggesting the occurrence of interactions that are not stable in the toeprint assay.     

Role of hypusinated eukaryotic translation initiation factor 5A in polyamine depletion-induced cytostasis

We have earlier shown that alpha-methylated spermidine and spermine analogues rescue cells from polyamine depletion-induced growth inhibition and maintain pancreatic integrity under severe polyamine deprivation. However, because alpha-methylspermidine can serve as a precursor of hypusine, an integral part of functional eukaryotic translation initiation factor 5A required for cell proliferation, and because alpha, omega-bismethylspermine can be converted to methylspermidine, it is not entirely clear whether the restoration of cell growth is actually attributable to hypusine formed from these polyamine analogues. Here, we have used optically active isomers of methylated spermidine and spermine and show that polyamine depletion-induced acute cytostasis in cultured cells could be reversed by all the isomers of the methylpolyamines irrespective of whether they served or not as precursors of hypusine. In transgenic rats with activated polyamine catabolism, all the isomers similarly restored liver regeneration and reduced plasma alpha-amylase activity associated with induced pancreatitis. Under the above experimental conditions, the (S, S)- but not the (R, R)-isomer of bismethylspermine was converted to methylspermidine apparently through the action of spermine oxidase strongly preferring the (S, S)-isomer. Of the analogues, however, only (S)-methylspermidine sustained cell growth during prolonged (more than 1 week) inhibition of polyamine biosynthesis. It was also the only isomer efficiently converted to hypusine, indicating that deoxyhypusine synthase likewise possesses hidden stereospecificity. Taken together, the results show that growth inhibition in response to polyamine depletion involves two phases, an acute and a late hypusine-dependent phase.