eIF2 Is a G-Protein Switch that Carries Met-tRNAi onto the Ribosome
The Met-tRNAi is delivered to the 40S subunit in the TC with eIF2·GTP. The affinity of Met-tRNAi is greater for eIF2·GTP than for eIF2·GDP, and this affinity switch depends on the methionine moiety on the Met-tRNAi (Kapp and Lorsch 2004). This contribution of methionine, plus the stimulatory role of the unique A1:U72 base pair (bp) in the acceptor stem of tRNAi in binding eIF2 (Kapp and Lorsch 2004; Pestova et al. 2007), presumably act to prevent binding of elongator tRNAs to the factor. This specificity, coupled with the requirement for eIF2 to load tRNA onto the 40S subunit, is thought to eliminate the need for a mechanism to reject elongator tRNAs during PIC assembly, a process in bacteria that relies heavily on IF3 (Hershey and Merrick 2000). (As described below, a structural homolog of IF3 is absent in eukaryotes, but eIF1 acts similarly to ensure selection of AUG as a start codon.) Understanding the structural basis for the stimulatory effects of methionine, the A1:U72 bp, and GTP versus GDP on initiator tRNA binding to eIF2 would be advanced by high-resolution structural analysis of the complete TC. Whereas the crystal structure of the archaeal ortholog (aIF2) has been solved, as well as various aIF2 subcomplexes bound to GDP or GTP analogs (reviewed in Schmitt et al. 2010), no crystal structures or cryo-EM (electron microscopy) models of heterotrimeric eIF2 have been described.
eIF2γ binds directly to both GTP and Met-tRNAi and it appears that the α and β subunits each increase the affinity of the eIF2 complex for Met-tRNAi by ∼100-fold (Naveau et al. 2010), but it is unknown whether this stimulatory effect involves direct contacts between Met-tRNAi and eIF2α or eIF2β. Based on the crystal structure of a heterotrimer of aIF2β, aIF2γ, and a portion of aIF2α (Yatime et al. 2007) it has been proposed that the α and β subunits allosterically induce a conformation in aIF2γ with high affinity for Met-tRNAi (Naveau et al. 2010). Evidence consistent with an allosteric mechanism, at least for eIF2α, comes from directed hydroxyl radical cleavage mapping of Met-tRNAi binding to yeast eIF2 in reconstituted PICs. Met-tRNAi was cleaved by free radicals generated from particular positions in eIF2γ or eIF2β, but not from eIF2α, suggesting the latter does not make direct contact with the tRNA. Interestingly, the patterns of cleavage imply a mode of initiator binding to eIF2γ dramatically different from that seen in crystal structures of the EF-Tu·GDPNP·Phe-tRNAPhe TC, which delivers aminoacylated tRNAs to the A site during elongation. In contrast to the latter complex, domain III of eIF2γ, the subunit homologous to EF-Tu, does not contact the T stem of Met-tRNAi; instead the sole contact is with the methionylated acceptor end of the tRNA in a pocket in eIF2γ formed between the G domain and domain II (Shin et al. 2011). A recent crystal structure of the TC formed by an archaeal aIF2, GDPNP, and E. coli Met-tRNA(fMet) also demonstrated that the tRNA is bound by aIF2 in a manner dramatically distinct from that of elongator tRNA binding to EF-Tu (Schmitt et al. 2012). Consistent with previous models (Schmitt et al. 2010; Shin et al. 2011), the acceptor end of the tRNA binds to aIF2γ according to the EF-Tu paradigm; however, the T-stem minor groove does not contact aIF2 and, instead, the T-loop in the tRNA “elbow” interacts with regions of the aIF2α subunit. As these last contacts were not detected in the hydroxyl radical probing of the eIF2 TC (Shin et al. 2011), it remains to be seen whether they are important in solution and conserved in eukaryotic TC.
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Importantly, the patterns of free radical-induced cleavages of 18S rRNA observed in this last study suggested that eIF2γ domain III interacts with h44 of 18S rRNA, but no other contacts between eIF2 and 18S rRNA were detected. Using the cleavage data to dock eIF2γ onto h44 and the 3′ end of Met-tRNAi, making use of high-resolution structures of a bacterial 70S·tRNA·mRNA complex (Selmer et al. 2006), the 40S·eIF1 complex (Rabl et al. 2011), and aIF2αγ and aIF2βγ heterodimers (Yatime et al. 2006, 2007) (among others), a structural model of the 43S PIC was constructed (Shin et al. 2011).
Although this model represents an important step, high-resolution crystal structures and cryo-EM reconstructions of free TC and TC bound to the 43S PIC remain critical goals. In addition, the model does not include known interactions of the eIF2β N-terminal domain (NTD) (lacking in aIF2β) with eIFs 1 and 5 in the MFC (Asano et al. 2000; Singh et al. 2004), and there might be contacts between eIF2α or eIF2β with 40S ribosomal proteins not detected by the hydroxyl radical mapping. Identifying mutations in yeast eIF2 subunits, ribosomal proteins, and 18S rRNA that reduce TC binding to the PIC should help identify the eIF2·40S contacts most critical in vivo. Only one such mutation has been identified in domain III of eIF2γ (R439A) and it produces a synergistic reduction in TC binding to reconstituted 43S·mRNA complexes when combined with an 18S rRNA substitution in helix 28 (A1152U) (Shin et al. 2011) that likely weakens interaction of the anticodon stem loop (ASL) of Met-tRNAi with the 40S P site (Dong et al. 2008). Identifying h44 mutations with these phenotypes would provide valuable support for the Shin et al. model of the 43S PIC.
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