Recognition of the 5'-end of eukaryotic mRNA by the ribosomal 43 S preinitiation complex involves the eukaryotic translation initiation factor eIF-4E (eIF-4alpha). Deletion mutants of the eIF-4E gene of Saccharomyces cerevisiae (CDC33) encoded proteins with reduced affinity for the 5'-cap. One of these mutant proteins lacked any detectable binding to a cap analogue binding column, yet was still able to support cell growth. More than 17% of the total eIF-4E amino acid sequence could be removed without fully inactivating this factor. At least 30 of the N-terminal amino acids are not essential for function. The minimal functional eIF-4E protein segment therefore comprises at most 176 amino acids. The translation and growth defects of the deletion mutants could be at least partially compensated by increases in eIF-4E synthesis, possibly due to a mass-action effect on mRNA binding. Electroporation of yeast spheroplasts with in vitro synthesized mRNA allowed us to characterize the ability of eIF-4E mutant strains to distinguish between capped and uncapped mRNAs in vivo. Our data show that the cap specificity of eIF-4E determines to what extent the translational apparatus differentiates between capped and uncapped mRNAs and indicate the minimum relative mRNA (cap) binding activity of eIF-4E required for yeast cell viability.

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Journal Article

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Vasilescu S, Ptushkina M, Linz B, Müller PP, McCarthy JE

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