tRNA^Gly and tRNA^Ala show discriminator base dichotomy in Bacteria. (a) Clover leaf model of tRNA with the discriminator base highlighted in red.
Distribution of the discriminator base in all tRNAs across the three domains of life.

Frequency distribution of tRNA acceptor stem elements across bacterial tRNAs, comparing and contrasting between tRNA^Gly and tRNA^Ala. Red circle indicates the discriminator base.

DTD avoids glycine misincorporation into proteins. (a) GFP-based fluorescence reporter assay for visualizing alanine-to-glycine mistranslation, wherein the mutant GFP G67A (65TYA67) will fluoresce only when TYA is mistranslated to TYG.

Model showing N73 dichotomy in bacterial tRNA^Gly and tRNA^Ala, enabling protection of the cognate Gly-tRNA^Gly (both proteinogenic and non-proteinogenic) predominantly by U73, while effecting efficient removal of the non-cognate Gly-tRNA^Ala (having A73 and G3.U70) to prevent alanine-to-glycine mistranslation.

ATD belongs to the DTD-like fold
Crystal structure of MmATD homodimer (blue) superimposed on that of PfDTD homodimer (cyan; PDB id: 4NBI)

Structural superposition of MmATD on PfDTD displaying the cross-subunit Gly-Pro motif

ATD has a Gly-transPro motif in the active site, unlike a Gly-cisPro motif in DTD

ATD is a unique and dedicated proofreading factor that rectifies a critical tRNA selection error. Model for mis-selection and consequent misacylation of tRNAThr(G4•U69) with L-alanine by AlaRSND, and its subsequent proofreading by ATD.

Mischarging Glycine and Serine by AlaRS

Misacylation of tRNA^Ala with glycine by AlaRS and its prevention/rectification by DTD.

DTD doubles as a key factor to uncouple glycine mischarged on tRNA^Ala.

Crystal structure of PfDTD with Gly3AA

Network of interactions of Gly3AA with active site residues of PfDTD. *Residues from the dimeric counterpart

Catalytic chamber in the active site of PfDTD can accommodate a water molecule

Model for protection of Gly-tRNA^Gly by EF-Tu