An important step in initiation of protein synthesis in Escherichia coli is the specific formylation of the initiator methionyl-tRNA (Met-tRNA) by Met-tRNA transformylase. The determinants for formylation are clustered mostly in the acceptor stem of the initiator tRNA. Here we use NMR spectroscopy to characterize the conformation of two RNA microhelices, which correspond to the acceptor stem of mutants of E. coli initiator tRNA and which differ only at the position corresponding to the "discriminator base" in tRNAs. One of the mutant tRNAs is an extremely poor substrate for Met-tRNA transformylase, whereas the other one is a much better substrate. We show that one microhelix forms a structure in which its 3'-ACCA sequence extends the stacking of the acceptor stem. The other microhelix forms a structure in which its 3'-UCCA sequence folds back such that the 3'-terminal A22 is in close proximity to G1. These results highlight the importance of the discriminator base in determining tRNA conformation at the 3' end. They also suggest a correlation between tRNA structure at the 3' end and its recognition by Met-tRNA transformylase.