Mechanisms and regulation of translation initiation

The eukaryotic translation initiation pathway produces an 80S ribosome bound to mRNA with methionyl initiator tRNA (Met-tRNA_i^Met) base-paired to the AUG start codon in the ribosomal P-site (Fig.1).

The Met-tRNA_i^Met is recruited to the small (40S) ribosomal subunit in a ternary complex (TC) with GTP-bound eIF2, to produce the 43S preinitiation complex (PIC). In vitro, this reaction is stimulated by eIF1, eIF1A, eIF3, and eIF5. The eIF3, eIF5, eIF1 and TC can be isolated in a multifactor complex (MFC) from budding yeast whose formation is thought to promote binding of all constituent factors to 40S subunits in vivo. The 43S PIC interacts with the 5’ end of mRNA in a manner stimulated by factors that bind to the m⁷G cap of the mRNA (eIFs 4E, -4G, -4A, -4B) or poly(A) tail (PABP), and by eIF3, to produce the 48S PIC. The PIC scans the leader until the anticodon of Met-tRNA_i^Met base-pairs with an AUG codon. Scanning to the AUG is promoted by eIF1, eIF1A and eIF4G. In the 48S PIC, the GTP bound to eIF2 is partially hydrolyzed to GDP and inorganic phosphate (Pi) in a manner stimulated by GTPase activating protein (GAP) eIF5. The recognition of AUG allows P_i release from eIF2•GDP•Pi to complete GTP hydrolysis by the TC. Met-tRNA_i^Met is released from eIF2-GDP into the P site, allowing subsequent joining of the 60S subunit catalyzed by eIF5B. The eIF2-GDP is recycled to eIF2-GTP by the guanine nucleotide exchange factor eIF2B for reassembly of the TC (reviewed in ^1,2).

Functions of the eIFs have been defined primarily by in vitro analyses of partial reactions of the initiation pathway using purified components from mammals or (more recently) budding yeast. While the yeast factors have critical functions in vivo, it is often unclear whether their in vitro activities correspond to their essential functions in living cells, and little is known about how these functions are carried out at the molecular level. Genetic and biochemical analysis of factors involved in the translational control of GCN4 has provided strong evidence that, in living cells, eIF2 is crucial for recruitment of Met-tRNA_i^Met to 40S ribosomes (ie. 43S assembly), that eIF1A and certain eIF3 subunits stimulate this reaction, and that the GEF eIF2B is required for optimum TC assembly. Phosphorylation of the a-subunit of eIF2 (eIF2[aP]) by GCN2 in starved cells impairs recycling of eIF2-GDP to eIF2-GTP by eIF2B. While reducing general translation, this specifically induces GCN4 translation by a reinitiation mechanism involving 4 uORFs in the mRNA leader (Fig. 2A).

After translating uORF1, ~50% of the 40S subunits resume scanning downstream in both starved and nonstarved cells. In nonstarved cells, all of the subunits quickly rebind TC, reinitiate at uORF4, and dissociate from the mRNA after translating uORF4. When TC levels are reduced by eIF2(aP), a fraction of ribosomes fails to rebind TC until scanning past uORF4, allowing them to reinitiate at GCN4 instead. Consistent with this, the bypass of uORF4 and induction of GCN4 translation in starved cells is suppressed by overproducing all three subunits of eIF2, or all four essential subunits of eIF2B, both conditions expected to elevate TC levels. Morever, GCN4 translation is constitutively derepressed (Gcd^- phenotype) in mutants with defects in eIF2 or eIF2B subunits, or Met-tRNA_i^Met biogenesis, in which TC assembly is impaired. More recently, we showed that truncating the eIF1A C-terminal tail (DC) or overproducing the N-terminal tail (NTT) of eIF3c/NIP1 (c/NIP1) also produce Gcd^- phenotypes that are diminished by overproducing all of the components of TC from a high-copy plasmid (hc-TC), suggesting that these mutations delay TC loading on 40S subunits scanning downstream from uORF1, allowing a fraction to bypass uORFs 2-4 and reinitiate at GCN4 in the absence of eIF2(aP) (Fig. 2B) ¹.

The molecular mechanism of ribosomal scanning and accurate AUG selection is also amenable to genetic and biochemical analyses using yeast.  Genetic studies established that tRNA_i^Met, the subunits of eIF2, eIF1 and eIF5 all contribute to stringent selection of AUG as start codon during scanning, as mutations in these factors confer a Sui^- (suppressor of initiation codon) phenotype signifying increased initiation at UUG start codons. This is recognized by suppression of the histidine requirement conferred by a mutation (the his4-303)in the start codon of the histidine biosynthetic gene HIS4 resulting from aberrant initiation at an in-frame UUG codon (Fig.5).

Previous biochemical analysis of Sui^- mutations in eIF2 subunits and eIF5 by Donahue and colleagues suggested that the rate of eIF5-catalyzed GTP hydrolysis by TC and dissociation of Met-tRNA_i^Met from eIF2-GDP are key determinants of stringent AUG selection ³. Recently, we identified Sui^- mutations, and also (Ssu^-) mutations that suppress UUG initiation, in eIF1A, implicating this factor in scanning and AUG selection. As scanning is fundamental to GCN4 translational control, isolation of mutations impairing induction of GCN4 (Gcn^- phenotype) in eIF5, eIF1A and eIF3 has implicated domains/residues of these factors in scanning or AUG recognition as well ¹. Recent genetic and biochemical studies suggest that functional interactions between eIF1A and eIF5 in the PIC regulate scanning ⁴, and also that eIF1 plays a “gatekeeper” function in preventing non-AUG selection ⁵, by promoting scanning ⁶ and blocking P_i release from eIF2-GDP-P_i ⁷ until AUG enters the P-site (Fig. 6).

a. Preinitiation complex assembly

b. Scanning and AUG selection