THE A1-CENTER-DOT-U72 BASE-PAIR CONSERVED IN EUKARYOTIC INITIATOR TRANSFER-RNAS IS IMPORTANT SPECIFICALLY FOR BINDING TO THE EUKARYOTIC TRANSLATION INITIATION-FACTOR EIF2

The formation of a specific ternary complex between eukaryotic initiat ion factor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation initiation in the cytoplasmic protei n-synthesizing system of eukaryotes. We show that the Al . U72 base pa ir conserved at the end of the acceptor stem in eukaryotic and archaeb acterial initiator methionine tRNAs plays an important role in this in teraction. We changed the Al . U72 base pair of the human initiator tR NA to G1 . C72 and expressed the wild-type and mutant tRNA genes in th e yeast Saccharomyces cerevisiae by using constructs previously develo ped in our laboratory for expression of the human initiator tRNA gene in yeasts. We show that both the wild-type and mutant human initiator tRNAs are aminoacylated well in vivo. We have isolated the wild-type a nd mutant human initiator tRNAs in substantially pure form, free of th e yeast initiator tRNA, and have analyzed their properties in vitro. T he G1 . C72 mutation affects specifically the binding affinity of eIF2 for the initiator tRNA. It has no effect on the subsequent formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the puromycin reactivity of the Met-tRNA in the 80S initia tion complex.