The eIF1A C-terminal domain promotes initiation complex assembly, scanning and AUG selection in vivo

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Abstract
Translation initiation factor 1A stimulates 40S‐binding of the eukaryotic initiation factor 2 (eIF2)/GTP/Met‐tRNAiMet ternary complex (TC) and promotes scanning in vitro . eIF1A contains an OB‐fold present in bacterial IF1 plus N‐ and C‐terminal extensions. Truncating the C‐terminus (Δ C ) or mutating OB‐fold residues ( 66–70 ) of eIF1A reduced general translation in vivo but increased GCN4 translation (Gcd− phenotype) in a manner suppressed by overexpressing TC. Consistent with this, both mutations diminished 40S‐bound TC, eIF5 and eIF3 in vivo , and Δ C impaired TC recruitment in vitro . The assembly defects of the OB‐fold mutation can be attributed to reduced 40S‐binding of eIF1A, whereas Δ C impairs eIF1A function on the ribosome. A substitution in the C‐terminal helix ( 98 – 101 ) also reduced 43S assembly in vivo . Rather than producing a Gcd− phenotype, however, 98 – 101 impairs GCN4 derepression in a manner consistent with defective scanning by reinitiating ribosomes. Indeed, 98 – 101 allows formation of aberrant 48S complexes in vitro and increases utilization of non‐AUG codons in vivo . Thus, the OB‐fold is crucial for ribosome‐binding and the C‐terminal domain of eIF1A has eukaryotic‐specific functions in TC recruitment and scanning.