The acceptor stem domain of tRNAAla contains the major recognition elements for aminoacylation by Escherichia coli alanine-tRNA synthetase (AlaRS). Previous studies established that a simple base pair transversion (G1:C72 → C1:G72) completely abolishes in vitro aminoacylation with alanine of duplexAla substrates. Subsequent atomic group "mutagenesis" experiments, wherein over 30 base pair variants were introduced at this site, revealed that the presence of a major groove carbonyl oxygen at position 72 functions as a negative determinant. Although, these previous biochemical studies strongly suggested the critical nature of a blocking element at this site, they could not rule out the possibility that conformational changes were responsible for the observed effects on aminoacylation. The mechanism of discrimination at the first base pair is further examined here. In particular, to establish whether conformational change contributes to the striking difference in aminoacylation activity, NMR spectroscopy and molecular dynamics (MD) simulations were carried out. The solution NMR assignments indicate that the global conformations of the wild-type microhelix versus the C1:G72-containing variant are very similar. This is consistent with the predicted structures based on MD simulations; on the basis of average structures and dynamical analysis of trajectories, there are no statistically significant differences between the variant and the wild-type microhelices with respect to either helical structure or A73 stacking interactions. Thus, AlaRS is indeed sensitive to specific atomic groups in the inactive C1 :G72 variant rather than to conformational changes associated with this base pair transversion.