Two GTPases are required for translation initiation: eIF2 and eIF5B. eIF2 forms a ternary complex with GTP and Met-tRNA and binds Met-tRNA to the 40S ribosome in the first step of translation initiation. eIF5B promotes subunit joining in the final step of protein synthesis. We previously obtained the crystal structures of both the GTP-binding eIF2g subunit and eIF5B. Subsequently, we have used molecular genetic structure-function analyses to dissect the functional properties of these factors. Our latest findings include dissection of the role of the eIF2 GTP-binding domain in start codon selection and using genetic and physical approaches to map the eIF5B–ribosome interface.
The translation initiation factor eIF2 binds the initiator Met-tRNA to the small ribosomal subunit. To gain further insights into the role of GTP binding and hydrolysis by eIF2, we mutated a conserved residue in the eIF2γ GTP-binding domain Switch I element. The mutation impaired Met-tRNA binding and enhanced initiation from a non-canonical UUG codon. Second site suppressors of the Switch I element mutation restored Met-tRNA binding but had differing impacts on UUG initiation. This uncoupling of start codon selection and Met-tRNA binding affinity to eIF2 indicates a more direct role for eIF2 in start site recognition than previously appreciated. Interestingly, overexpression of eIF1, which is thought to monitor codon-anticodon interaction during translation initiation, suppressed initiation at UUG codons in the eIF2γ mutants. We propose that structural alterations in eIF2γ subtly alter the conformation of Met-tRNA on the 40S subunit and thereby affect the fidelity of start codon recognition independent of Met-tRNA binding affinity (Alone et al. MCB 2008).
The GTP-binding protein eIF5B catalyzes ribosomal subunit joining in the final step of translation initiation. Our previous studies revealed that GTP hydrolysis by eIF5B activates a regulatory switch required for eIF5B release from the ribosome following subunit joining. Mutations in eIF5B that impair GTP hydrolysis impair yeast cell growth due to failure to dissociate the factor from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA within the body of the small ribosomal subunit as well as mutations in domain II of eIF5B suppressed the toxic effects of the GTPase-deficient mutants of eIF5B. Interestingly, hydroxyl radical mapping experiments revealed that domain II of eIF5B docks on the ribosome in the vicinity of helix h5. Additional studies showed that helix h5 is also important for the function of the translation elongation factor GTP-binding protein eEF2. Thus, these studies provide in vivo evidence supporting a functionally important docking of domain II of translational GTPases on the body of the small ribosomal subunit (Shin et al. MCB 2009).
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