[
International Worm Meeting,
2009]
Recognition of AUG start codon during translation initiation is critical for decoding the genetic information on mRNAs. In prokaryotes, the Shine-Dalgarno sequence located 5'' to the AUG on the mRNA complements to a region on the 16S rRNA and this base-pairing interaction positions the mRNA so that the initiator tRNA finds the AUG efficiently. In contrast, there is no evidence in eukaryotes that such base-paring occurs, and instead eukaryotes appear to rely mainly on the base pairing of the AUG with the anticodon of the initiator tRNA. It is postulated that recognition is achieved through ribosomal scanning, in which a pre-initiation complex, consisting of the small ribosomal subunit, multiple translation initiation factors (eIFs) and initiator tRNA, moves along the mRNA to examine each codon by base pairing with the anticodon. Perfect base pairing triggers the hydrolysis of GTP by the heterotrimeric G-protein eIF2 and leads to a cascade of molecular interactions that allow translation to start. We have developed a genetic system in C. elegans to study how the start codon AUG is recognized. Our strategy is similar to that employed in the isolation of S. cerevisiae sui (suppressor of initiation codon) mutants, whereby a selectable reporter that contained an altered initiation codon was utilized (Donahue et al., Cell 54:621, 1988). We changed the ATG translation initiation codon of an abundantly expressed GFP reporter to that of a GTG codon and found that the GFP signal is almost completely eliminated in wild type worms. With this silent reporter, we performed genetic screens and obtained mutants that showed varying degrees of GFP expression. Mutations in two mutant strains have been identified. Both are missense mutations; one in the b subunit of eIF2 and the other in eIF1. We further studied if this newly identified eIF2b mutation and the yeast SUI3 mutations when introduced into the corresponding sites of the C. elegans gene could initiation translation with other non-AUG codons. We found that all of these mutants allow GFP to be synthesized from reporters that contain altered start site codons that differ by one base either in the first or third base position. Changes in the second base position result in much less GFP expression and changes of two or three bases reduce expression to undetectable levels. Our results differ from what was previously observed in S. cerevisiae. The SUI3 mutations only allow translation from the non-AUG codon UUG in the yeast reporter assay (Huang et al., Genes Dev 11:2396, 1997). Our analyses in C. elegans suggest that eIF2b plays a role in recognizing proper codon-anticodon pairing and/or in relaying the signal of correct base pairing which leads to GTP hydrolysis.