Abstract: We demonstrate here, using RNA variants derived from tRNAAsp, that the minimalist aminoacylated structure able to interact efficiently with elongation factor Tu comprises a 10 base-pair helix linked to the 3'-terminal NCCA sequence. Shorter structures can interact with the elongation factor, but with significantly decreased affinity. Conserved features in the aminoacyl acceptor branch of tRNAs, such as base pair G53-C61 and the T-loop architecture, could be replaced respectively by the inverted base pair C53-G61 and by unusual anticodon loop or tetraloop sequences. Variants of whole tRNAAsp or of the 12 base-pair aspartate minihelix, with enlarged 13 base-pair long aminoacyl acceptor branches, as in selenocysteine-inserting tRNAs that are not recognized by elongation factor Tu, keep their binding ability to this factor. These functional results are well accounted for by the crystallographic structure of the Thermus thermophilus binary EF-Tu.GTP complex, which possesses a binding cleft accommodating the minimalist 10 base-pair domain of the tRNA aminoacyl acceptor branch.