A discriminator code-based DTD surveillance ensures faithful glycine delivery for protein biosynthesis in bacteria.

D-aminoacyl-tRNA deacylase (DTD) acts on achiral glycine, in addition to D-amino acids, attached to tRNA. We have recently shown that this activity enables DTD to clear noncognate Gly-tRNA(Ala) with 1000-fold higher efficiency than its activity on Gly-tRNA(Gly), indicating tRNA-based modulation of DTD (Pawar et al., 2017). Here, we show that tRNA's discriminator base predominantly accounts for this activity difference and is the key to selection by DTD. Accordingly, the uracil discriminator base, serving as a negative determinant, prevents Gly-tRNA(Gly) misediting by DTD and this protection is augmented by EF-Tu. Intriguingly, eukaryotic DTD has inverted discriminator base specificity and uses only G3.U70 for tRNA(Gly/Ala) discrimination. Moreover, DTD prevents alanine-to-glycine misincorporation in proteins rather than only recycling mischarged tRNA(Ala). Overall, the study reveals the unique co-evolution of DTD and discriminator base, and suggests DTD's strong selection pressure on bacterial tRNA(Gly)s to retain a pyrimidine discriminator code.