We measured kinetic parameters in vitro and directly analyzed aminoacylation and formylation levels in vivo to study recognition of Escherichia coli initiator tRNA mutants by E. coli Met-tRNA synthetase and Met-tRNA transformylase. We show that, in addition to the anticodon sequence, mutations in the "discriminator" base A73 also affect aminoacylation. An A73----U change has a small effect, but a change to G73 or C73 significantly lowers Vmax/Kappm for in vitro aminoacylation and leads to appreciable accumulation of uncharged tRNA in vivo. Significantly, coupling of the G73 mutation with G72, a neighboring-base mutation, results in a tRNA essentially uncharged in vivo. Coupling of C73 and U73 mutations with G72 does not have such an effect. Elements crucial for Met-tRNA transformylase recognition of tRNAs are located at the end of the acceptor stem. These elements include a weak base pair or a mismatch between nucleotides (nt) 1 and 72 and base pairs 2.71 and 3.70. The natures of nt 1 and 72 are less important than the fact that they do not form a strong Watson-Crick base pair. Interestingly, the negative effect of a C.G base pair between nt 1 and 72 is suppressed by mutation of the neighboring nucleotide A73 to either C73 or U73. The presence of C73 or U73 could destabilize the C1.G72 base pair at the end of an RNA helix. Thus, in some tRNAs, the discriminator base could affect stability of the base pair between nt 1 and 72 and thereby the structure of tRNA at the end of the acceptor stem.