Bacillus cereus 14579 encodes two tRNAs with the CCA anticodon, tRNA(Trp) and tRNA(Other). tRNA(Trp) was separately aminoacylated by two enzymes, TrpRS1 and TrpRS2, which share only 34% similarity and display different catalytic capacities and specificities. TrpRS1 was 18-fold more proficient at aminoacylating tRNA(Trp) with Trp, while TrpRS2 more efficiently utilizes the Trp analog 5-hydroxy Trp. tRNA(Other) was not aminoacylated by either TrpRS but instead by the combined activity of LysRS1 and LysRS2, which recognized sequence elements absent from tRNA(Trp). Polysomes were found to contain tRNA(Trp), consistent with its role in translation, but not tRNA(Other) suggesting a function outside protein synthesis. Regulation of the genes encoding TrpRS1 and TrpRS2 (trpS1 and trpS2) is dependent on riboswitch-mediated recognition of the CCA anticodon, and the role of tRNA(Other) in this process was investigated. Deletion of tRNA(Other) led to up to a 50 fold drop in trpS1 expression, which resulted in the loss of differential regulation of the trpS1 and trpS2 genes in stationary phase. These findings reveal that sequence-specific interactions with a tRNA anticodon can be confined to processes outside translation, suggesting a means by which such RNAs may evolve non-coding functions.