LIGAND INTERACTIONS WITH EUKARYOTIC TRANSLATION INITIATION-FACTOR-2 -ROLE OF THE GAMMA-SUBUNIT

Eukaryotic translation initiation factor 2 (eIF-2) comprises three non -identical subunits alpha, beta and gamma. In vitro, eIF-2 binds the i nitiator methionyl-tRNA in a GTP-dependent fashion. Based on similarit ies between eukaryotic eIF-2y proteins and eubacterial EF-Tu proteins, we previously proposed a major role for the gamma-subunit in binding guanine nucleotide and tRNA. We have tested this hypothesis by examini ng the biochemical activities of yeast eIF-2 purified from wildtype st rains and strains harboring mutations in the eIF-2y structural gene (G CD11) predicted to alter ligand binding by elF-2. The alteration of ty rosine 142 in yeast eIF-2y, corresponding to histidine 66 in Escherich ia coli EF-Tu, dramatically reduced the affinity of eIF-2 for Met-tRNA (i)(Met) without affecting the k(off) value for guanine nucleotides. I n contrast, nonlethal substitutions at a conserved lysine residue (K25 0) in the putative guanine ring-binding loop increased the off-rate fo r GDP, thereby mimicking the function of the guanine nucleotide exchan ge factor eIF-2B, without altering the apparent dissociation constant for Met-tRNA(i)(Met). For eIF-2[gamma-K250R], the increased off-rate a lso seen for GTP was masked by the presence of Met-tRNA(i)(Met) in vit ro. In vivo, increasing the dose of the yeast initiator tRNA gene supp ressed the slow-growth phenotype and reduced GCN4 expression in gcd11- K250R and gcd11-Y142H strains. These studies indicate that the gamma-s ubunit of eIF-2 does indeed provide EF-Tu-like function to the eIF-2 c omplex, and further suggest that the level of Met-tRNA(i)(Met) is crit ical for maintaining wild-type rates of initiation in vivo.