An operational RNA code relates specific amino acids to sequences/structures in RNA hairpin helices which reconstruct the seven-base-pair acceptor stems of transfer RNAs. These RNA oligonucleotides are aminoacylated by aminoacyl tRNA synthetases. The specificity and efficiency of aminoacylation are generally determined by three or four nucleotides which are near the site of amino acid attachment. These specificity-determining nucleotides include the so-called "discriminator base" and one or two base pairs within the first four base pairs of the helix. With three examples considered here, nucleotide sequence variations between the eubacterial E. coli tRNA acceptor stems and their human cytoplasmic and mitochondrial counterparts are shown to include changes of some of the nucleotides known to be essential for aminoacylation by the cognate E. coli enzymes. If the general locations of the specificity-determining nucleotides are the same in E. coli and human RNAs, these RNA sequence variations imply a similar covariation in sequences/structures of the E. coli and human tRNA synthetases. These covariations would reflect the integral relationship between the operational RNA code and the design and evolution of tRNA synthetases.