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Escherichia coli leucyl-tRNA synthetase (LeuRS) aminoacylates up to six different class II tRNA^leu molecules. Each has a distinct anticodon and varied nucleotides in other regions of the tRNA. Attempts to construct a minihelix RNA that can be aminoacylated with leucine have been unsuccessful. Herein, we describe the smallest tRNA^leu analog that has been aminoacylated to a significant extent to date. A series of tRNA^leu analogs with various domains and combinations of domains deleted was constructed. The minimal RNA that was efficiently aminoacylated with LeuRS was one in which the anticodon stem–loop and variable arm stem–loop, but neither the D-arm nor Tarm, were deleted. Aminoacylation of this minimal RNA was abolished when the discriminator base A73 was replaced with C73 or when putative tertiary interactions between the D-loop and T-loop were disrupted, suggesting that these …