Translocation within the acceptor helix of a major tRNA identity determinant

The genetic code is defined by the specific aminoacylations of tRNAs by ami noacyl-tRNA synthetases. Although the synthetases are widely conserved thro ugh evolution, aminoacylation of a given tRNA is often system specific-a sy nthetase from one source will not acylate its cognate tRNA from another. Th is system specificity is due commonly to variations in the sequence of a cr itical tRNA identity element. In bacteria and the cytoplasm of eukaryotes, an acceptor stem G3:U70 base pair marks a tRNA for aminoacylation with alan ine. In contrast, Drosophila melanogaster (Dm) mitochondrial (mt) tRNA(Ala) has a G2:U71 but not a G3:U70 pair. Here we show that this translocated GX and the adjacent G3:C70 are major determinants for recognition by Dm mt al anyl-tRNA synthetase (AlaRS). Additionally, G:U at the 3:70 position serves as an anti-determinant for Dm mt AlaRS. Consequently, the mitochondrial en zyme cannot charge cytoplasmic tRNA(Ala). All insect mitochondrial AlaRSs a ppear to have split apart recognition of mitochondrial from cytoplasmic tRN A(Ala) by translocation of G:U. This split may be essential for preventing introduction of ambiguous states into the genetic code.