Mutations in a GTP-binding motif of eukaryotic elongation factor 1A reduceboth translational fidelity and the requirement for nucleotide exchange

A series of mutations in the highly conserved N(153)KMD(156)GTP-binding mot if of the Saccharomyces cerevisiae translation elongation factor 1A (eEF1A) affect the GTP-dependent functions of the protein and increase misincorpor ation of amino acids in vitro. Two critical regulatory processes of transla tion elongation, guanine nucleotide exchange and translational fidelity, we re analyzed in strains with the N153T, D156N, and N153T/D156E mutations. Th ese strains are omnipotent suppressors of nonsense mutations, indicating re duced A site fidelity, which correlates with changes either in total transl ation rates in vivo or in GTPase activity in vitro, All three mutant protei ns also show an increase in the K-m for GTP. An in vivo system lacking the guanine nucleotide exchange factor eukaryotic elongation factor 1B alpha (e EF1B alpha) and supported for growth by excess eEF1A was used to show the t wo mutations with the highest K-m for GTP restore most but not all growth d efects found in these eEF1Ba deficient-strains to near wild type. An increa se in K-m alone, however, is not sufficient for suppression and may indicat e eEF1B alpha performs additional functions. Additionally, eEF1A mutations that suppress the requirement for guanine nucleotide exchange may not effec tively perform all the functions of eEF1A in vivo.