Summary The RNA helicase eIF4A plays a key role in unwinding of mRNA and scanning during translation initiation. Free eIF4A is a poor helicase and requires the accessory proteins eIF4G and eIF4H. However, the structure of the helicase complex and the mechanisms of stimulation of eIF4A activity have remained elusive. Here we report the topology of the eIF4A/4G/4H helicase complex, which is built from multiple experimentally observed domain-domain contacts. Remarkably, some of the interactions are continuously rearranged during the ATP binding/hydrolysis cycle of the helicase. We show that the accessory proteins modulate the affinity of eIF4A for ATP by interacting simultaneously with both helicase domains and promoting either the closed, ATP-bound conformation or the open, nucleotide-free conformation. The topology of the complex and the spatial arrangement of the RNA-binding surfaces offer insights into their roles in stimulation of helicase activity and the mechanisms of mRNA unwinding and scanning.

Translation, the process of mRNA-encoded protein synthesis, requires a complex apparatus, composed of the ribosome, tRNAs and additional protein factors, including aminoacyl tRNA synthetases. The ribosome provides the platform for proper assembly of mRNA, tRNAs and protein factors and carries the peptidyl-transferase activity. It consists of small and large subunits. The ribosomes are ribonucleoprotein particles with a ribosomal RNA core, to which multiple ribosomal proteins are bound. The sequence and structure of ribosomal RNAs, tRNAs, some of the ribosomal proteins and some of the additional protein factors are conserved in all kingdoms, underlying the common origin of the translation apparatus. Translation can be subdivided into several steps: initiation, elongation, termination and recycling. Of these, initiation is the most complex and the most divergent among the different kingdoms of life. A great amount of new structural, biochemical and genetic information on translation initiation has been accumulated in recent years, which led to the realization that initiation also shows a great degree of conservation throughout evolution. In this review, we summarize the available structural and functional data on translation initiation in the context of evolution, drawing parallels between eubacteria, archaea, and eukaryotes. We will start with an overview of the ribosome structure and of translation in general, placing emphasis on factors and processes with relevance to initiation. The major steps in initiation and the factors involved will be described, followed by discussion of the structure and function of the individual initiation factors throughout evolution. We will conclude with a summary of the available information on the kinetic and thermodynamic aspects of translation initiation.

Eukaryotic initiation factor (eIF) eIF1 maintains the fidelity of initiation codon selection by enabling 43S complexes to reject codon-anticodon mismatches, to recognize the initiation codon context, and to discriminate against establishing a codon-anticodon interaction with AUGs located <8 nt from the 5-end of mRNA. To understand how eIF1 plays its discriminatory role, we determined its position on the 40S ribosomal subunit using directed hydroxyl radical cleavage. The cleavage of 18S rRNA in helices 23b, 24a, and 44 by hydroxyl radicals generated from Fe(II) tethered to seven positions on the surface of eIF1 places eIF1 on the interface surface of the platform of the 40S subunit in the proximity of the ribosomal P-site. The position of eIF1 on the 40S subunit suggests that although eIF1 is unable to inspect the region of initiation codon directly, its position close to the P-site is very favorable for an indirect mechanism of eIF1's action by influencing the conformation of the platform of the 40S subunit and the positions of mRNA and initiator tRNA in initiation complexes. Unexpectedly, the position of eIF1 on the 40S subunit was strikingly similar to the position on the 30S ribosomal subunit of the sequence and structurally unrelated C-terminal domain of prokaryotic initiation factor IF3, which also participates in initiation codon selection in prokaryotes.

The eukaryotic translation initiation factors 4A (eIF4A) and 4G (eIF4G) are crucial for the assembly of the translationally active ribosome. Together with eIF4E, they form the eIF4F complex, which recruits the 40S subunit to the 5 cap of mRNA. The two-domain RNA helicase eIF4A is a very weak helicase by itself, but the activity is enhanced upon interaction with the scaffolding protein eIF4G. Here we show that, albeit both eIF4A domains play a role in binding the middle domain of eIF4G (eIF4G-m, amino acids 745-1003), the main interaction surface is located on the C-terminal domain. We use NMR spectroscopy to define the binding site and find that the contact surface is adjacent to the RNA-, ATP-, and eIF4A-NTD-interacting regions. Mutations of interface residues abrogated binding, confirmed the interface, and showed that the N-terminal end of eIF4G-m interacts with the C-terminal domain of eIF4A. The data suggest that eIF4G-m forms a soft clamp to stabilize the closed interdomain orientation of eIF4A. This model can explain the cooperativity between all binding partners of eIF4A (eIF4G, RNA, ATP) and stimulation of eIF4A activity in the eIF4F complex.[Keywords: Translation; protein-protein interaction; eIF4A; eIF4G; helicase; NMR] Supplementary material is available at http://www.genesdev.org.

SUMMARY Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs) eIF1, eIF1A, eIF2 and eIF5. The carboxyl-terminal domain (CTD) of eIF5 stimulates 43S preinitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2β on eIF5-CTD and find that they are partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. By abrogating eIF5-CTD binding to eIF2β, genetic and biochemical evidence indicate that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC. This study provides mechanistic insight into the novel role of eIF5-CTD’s dynamic interplay with eIF1 and eIF2β in switching PICs from an open to closed state at start codons.

RNA helicases represent a large family of proteins implicated in many biological processes including ribosome biogenesis, splicing, translation and mRNA degradation. However, these proteins have little substrate specificity, making inhibition of selected helicases a challenging problem. The prototypical DEAD box RNA helicase, eIF4A, works in conjunction with other translation factors to prepare mRNA templates for ribosome recruitment during translation initiation. Herein, we provide insight into the selectivity of a small molecule inhibitor of eIF4A, hippuristanol. This coral-derived natural product binds to amino acids adjacent to, and overlapping with, two conserved motifs present in the carboxy-terminal domain of eIF4A. Mutagenesis of amino acids within this region allowed us to alter the hippuristanol-sensitivity of eIF4A and undertake structure/function studies. Our results provide an understanding into how selective targeting of RNA helicases for pharmacological intervention can be achieved.

The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNAiMet 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-tRNAiMet, 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-tRNAiMet 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-tRNAiMet, 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.

Programmed Cell Death 4 (PDCD4) is a protein known to bind eukaryotic initiation factor 4A (eIF4A), inhibit translation initiation, and act as a tumor suppressor. PDCD4 contains two C-terminal MA3 domains, which are thought to be responsible for its inhibitory function. Here, we analyze the structures and inhibitory functions of these two PDCD4 MA3 domains by x-ray crystallography, NMR, and surface plasmon resonance. We show that both MA3 domains are structurally and functionally very similar and bind specifically to the eIF4A N-terminal domain (eIF4A-NTD) using similar binding interfaces. We found that the PDCD4 MA3 domains compete with the eIF4G MA3 domain and RNA for eIF4A binding. Our data provide evidence that PDCD4 inhibits translation initiation by displacing eIF4G and RNA from eIF4A. The PDCD4 MA3 domains act synergistically to form a tighter and more stable complex with eIF4A, which explains the need for two tandem MA3 domains.apoptosis ͉ eIF4G ͉ protein ͉ NMR ͉ x-ray crystallography P DCD4 is a tumor-suppressor protein that is up-regulated on induction of apoptosis (1) and down-regulated in certain aggressive tumors (2). PDCD4 is controlled by protein kinase S6K1 and the ubiquitin ligase SCF ␤TRCP , and its degradation is necessary for efficient protein translation in vivo, which is a prerequisite for cell growth and, consequently, for cell proliferation (3).PDCD4 is known to bind two eukaryotic translation initiation factors eIF4A and eIF4G (4-6). eIF4A is an RNA helicase that works as a subunit of eIF4F, a complex composed of eIF4G and eIF4E. The helicase activity of eIF4A itself is weak but is enhanced upon binding to eIF4G (7,8). eIF4G has two independent binding sites for eIF4A (9), one in the conserved middle domain (eIF4G-m, HEAT1/MIF4G) ( Fig. 1), and the other in the adjacent second HEAT domain (eIF4G-MA3, HEAT2/MA3) (reviewed in ref. 10). NMR binding studies have shown that eIF4G-m interacts mainly with the C-terminal domain of eIF4A (eIF4A-CTD) (11), whereas eIF4G-MA3 binds to the N-terminal domain of eIF4A (eIF4A-NTD) and only weakly to eIF4A-CTD (A.M., C.S., K.A.E., and G.W., unpublished work). Mutation and deletion analysis indicates that the interaction of eIF4A with eIF4G-m is necessary for translation, whereas the interaction of eIF4A with eIF4G-c (eIF4G-MA3ϩHEAT3 domain) plays a modulatory role (12).PDCD4 contains two MA3 domains after an N-terminal segment of little known function (1). MA3 is a well conserved ␣-helical motif with typically 3-5 helical hairpins and is a subtype of HEAT domains. A single MA3 domain is found in eIF4G-c (eIF4G-MA3, Fig. 1), which has been reported to also bind eIF4A (10, 13). Recently, crystal and solution structures of mouse PDCD4's Cterminal MA3 domain (MA3-c) were reported (14, 15). MA3-c was shown to bind eIF4A, compete with eIF4G-c, and was sufficient to inhibit translation initiation. However, some questions remained. First, the function of the N-terminal MA3 domain (MA3-m) was unclear. Mutations of conserved amino acid residues in either MA3-c or ...