Mutation analysis of the Cys-X2-Cys-X19-Cys-X2-Cys motif in the β subunit of eukaryotic translation initiation factor 2

摘要

Recessive lethal mutations in the β subunit of eIF-2 that restore HIS4 expression in the absence of an AUG start codon were isolated from diploid Saccharomyces cerevisiae strains. DNA sequence analysis of these alleles and of eIF-2β suppressor alleles isolated from haploid strains, identified point mutations that altered one of six amino acids that map to a Cys-X2-Cys-X19-Cys-X2-Cys “zinc finger” motif and immediately adjacent residues. Five of the affected amino acids are identical in the human and yeast eIF-2β protein. Together with earlier studies (), these point mutations implicate the zinc finger domain of eIF-2β in start-site selection during the scanning process. We have supplemented the mutations obtained by genetic selection with an additional set of constructed mutations in this region. Our studies indicate that the cysteine residues and the intervening amino acids of this motif are essential for eIF-2β function in translation in itiation in vivo. However, the effects observed in cells containing a copy of eIF-2β with a deletion of this motif suggest that this mutated form is still able to associate with other components of the initiation complex, imparting defects on translation initiation. Thus, this motif may be re quired only for later events that lead to initiator codon recognition. Alterations in defined positions, as found in our suppressor alleles, could lead to recognition of non-AUG codons.