RNA hairpin minihelices that recreate the acceptor-T psi C stem of Escherichia coli alanine tRNA are charged specifically with alanine, provided that they encode the critical G3:U70 base pair that is the major determinant for the identity of an alanine tRNA. These model substrates were used to investigate the role in charging of N73, the unpaired nucleotide that is just three positions removed from the amino acid attachment site and which is sometimes referred to as the "discriminator" base. Previous work showed that, while substrates which encode G3:U70 are all charged by alanine tRNA synthetase regardless of the base at position 73, catalytic efficiency is substantially higher with substrates that have the wild-type A73. To identify a specific step in aminoacylation that is affected by substitutions of A73, we studied the single turnover charging of A73, U73, C73, and G73 minihelices, using preformed, enzyme-bound alanyl adenylate and saturating concentrations of the respective minihelices. Compared to the A73 substrate, the transfer of activated amino acid to bound RNA is sharply reduced for the substituted N73 minihelices. The low efficiency of transfer is not due to an abortive reaction in which the adenylate or a transiently charged RNA is hydrolyzed. Instead, under the conditions used, the active adenylate remains on the enzyme for extended periods and simply reacts slowly with bound N73 RNA. The results suggest that the nature of the discriminator base is a critical determinant of the transition state for the reaction of bound alanyl adenylate with RNA on the surface of the enzyme.