Role of D-aminoacyl-tRNA deacylase beyond chiral proofreading as a cellular defense against glycine mischarging by AlaRS.

Abstract

Strict L-chiral rejection through Gly-<i>cis</i>Pro motif during chiral proofreading underlies the inability of D-aminoacyl-tRNA deacylase (DTD) to discriminate between D-amino acids and achiral glycine. The consequent Gly-tRNA^Gly 'misediting paradox' is resolved by EF-Tu in the cell. Here, we show that DTD's active site architecture can efficiently edit mischarged Gly-tRNA^Ala species four orders of magnitude more efficiently than even AlaRS, the only ubiquitous cellular checkpoint known for clearing the error. Also, DTD knockout in AlaRS editing-defective background causes pronounced toxicity in <i>Escherichia coli</i> even at low-glycine levels which is alleviated by alanine supplementation. We further demonstrate that DTD positively selects the universally invariant tRNA^Ala-specific G3•U70. Moreover, DTD's activity on non-cognate Gly-tRNA^Ala is conserved across all bacteria and eukaryotes, suggesting DTD's key cellular role as a glycine deacylator. Our study thus reveals a hitherto unknown function of DTD in cracking the universal mechanistic dilemma encountered by AlaRS, and its physiological importance.