No abstract

eIF1A is the eukaryotic ortholog of bacterial translation initiation factor IF1, but contains a helical domain and long unstructured N-terminal tail (NTT) and C-terminal tail (CTT) absent in IF1. Here, we identify elements in these accessory regions of eIF1A with dual functions in binding methionyl initiator tRNA (Met-tRNA i Met ) to the ribosome and in selecting AUG codons. A pair of repeats in the eIF1A CTT, dubbed Scanning Enhancer 1 (SE 1 ) and SE 2 , was found to stimulate recruitment of Met-tRNA i Met in the ternary complex (TC) with eIF2ÁGTP and also to block initiation at UUG codons. In contrast, the NTT and segments of the helical domain are required for the elevated UUG initiation occurring in SE mutants, and both regions also impede TC recruitment. Remarkably, mutations in these latter elements, dubbed scanning inhibitors SI 1 and SI 2 , reverse the defects in TC loading and UUG initiation conferred by SE substitutions, showing that the dual functions of SE elements in TC binding and UUG suppression are mechanistically linked. It appears that SE elements enhance TC binding in a conformation conducive to scanning but incompatible with initiation, whereas SI elements destabilize this conformation to enable full accommodation of Met-tRNA i Met in the P site for AUG selection.[Keywords: Translation; initiation; eIF1A; eIF2; initiator; scanning]Supplemental 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.

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

Background: Start codon recognition triggers eIF1 and P i release from the preinitiation complex. Results: The C-terminal tail of eIF1A moves closer to eIF5 upon start codon recognition, and this movement is required for P i release. Conclusion: eIF1 release and movement of the eIF1A C-terminal tail toward eIF5 are coupled processes. Significance: Start codon recognition induces coordinated movements of initiation factors that trigger downstream events.

In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNAi in a ‘PIN’ state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a yeast 48S pre-initiation complex (PIC), at an overall resolution of 3.0 Å, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG codonsin vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection.

The reticuloendothelial system plays a major role in iron metabolism. Despite this, the manner in which macrophages handle iron remains poorly understood. Mammalian cells utilize transferrin-dependent mechanisms to acquire iron via transferrin receptors 1 and 2 (TfR1 and TfR2) by receptor-mediated endocytosis. Here, we show for the first time that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is localized on human and murine macrophage cell surface. The expression of this surface GAPDH is regulated by the availability of iron in the medium. We further demonstrate that this GAPDH interacts with transferrin and the GAPDHtransferrin complex is subsequently internalized into the early endosomes. Our work sheds new light on the mechanisms involved in regulation of iron, vital for controlling numerous diseases and maintaining normal immune function. Thus, we propose an entirely new avenue for investigation with respect to transferrin uptake and regulation mechanisms in macrophages.Iron is an essential nutrient for all organisms as a constituent of hemoproteins and iron-sulfur proteins. In addition it is also a critical component of functional groups of several proteins involved in vital housekeeping functions. Cells of the immune system require iron for their normal functions such as proliferation, activation, and maturation of lymphocytes (1-5). Iron is also essential for macrophage-mediated cytotoxicity by the production of highly toxic hydroxyl radicals (6, 7). The mononuclear phagocyte system is composed of monocytes, macrophages, and their precursor cells, which play a vital role in iron metabolism by removing effete erythrocytes and recycling iron. These cells also acquire iron via the receptor-mediated uptake of transferrin and the hemoglobin scavenger receptor (8). Practically all extracellular iron circulating in the plasma is bound to transferrin, an abundant protein with high affinity for iron. Two mammalian transferrin receptors TfR1 4 and TfR2 have so far been characterized. Both these receptors are cell surface transmembrane, glycoproteins (9). Unlike TfR1, TfR2 is not regulated by intracellular iron concentrations. This receptor also binds transferrin in manner similar to TfR1, but with a 25-fold lower affinity (10,11,12). Iron uptake from transferrin involves binding to its receptors followed by internalization to the early endosomes. (13, 14). Although TfR-mediated iron uptake is the major pathway for iron acquisition, several studies have indicated that additional mechanisms independent of known TfRs exist; however, these have not been well characterized (15-18). GAPDH was previously considered to be an abundantly present cytosolic protein with a key role in energy metabolism. However, recent evidence has proved that it functions as a moonlighting protein in both prokaryotic and eukaryotic cells, often differentially localized within the cell (19 -21). It is of interest to note that in Staphylococcus aureus, cell wall-associated GAPDH had previously been identified as a trans...

SummaryLittle 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 pre-initiation complex (PIC) after start codon recognition to the final 80S initiation complex (IC). The resulting framework reveals that eIF5B 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 IC 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.