Abstract
Strict L-chiral rejection through Gly-cisPro 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-tRNAGly 'misediting paradox' is resolved by EF-Tu in the cell. Here, we show that DTD's active site architecture can efficiently edit mischarged Gly-tRNAAla 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 Escherichia coli even at low-glycine levels which is alleviated by alanine supplementation. We further demonstrate that DTD positively selects the universally invariant tRNAAla-specific G3•U70. Moreover, DTD's activity on non-cognate Gly-tRNAAla 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.
Highlights
D-aminoacyl-tRNA deacylase (DTD) is a key factor that keeps chiral errors away from the translational machinery by allowing only L-amino acids to form proteins and has been implicated in perpetuation of homochirality in the protein world (Calendar and Berg, 1967; Soutourina et al, 1999, 2000)
While occasional chiral errors that occur during aminoacylation are cleared by DTD to ensure the accuracy of aminoacyl-tRNAs present in the cellular pool, a major role is played by editing functions associated with about half of the 20 aminoacyl-tRNA synthetases to rectify the incorrect pairing of a similar non-cognate L-amino acid with a tRNA (Guo and Schimmel, 2012; Ibba and Soll, 2000)
We checked the accumulation of Gly-tRNAAla when alanyl-tRNA synthetase (AlaRS) editing was compromised, and it was found to be significantly high, almost equal to the level of AlatRNAAla formation (Figure 2—figure supplement 1)
Summary
D-aminoacyl-tRNA deacylase (DTD) is a key factor that keeps chiral errors away from the translational machinery by allowing only L-amino acids to form proteins and has been implicated in perpetuation of homochirality in the protein world (Calendar and Berg, 1967; Soutourina et al, 1999, 2000). While occasional chiral errors that occur during aminoacylation are cleared by DTD to ensure the accuracy of aminoacyl-tRNAs present in the cellular pool, a major role is played by editing functions associated with about half of the 20 aminoacyl-tRNA synthetases (aaRSs) to rectify the incorrect pairing of a similar non-cognate L-amino acid with a tRNA (Guo and Schimmel, 2012; Ibba and Soll, 2000). These aaRSs can edit the non-cognate amino acid at the aminoacylation site itself after the amino acid has been activated (i.e. formation of aminoacyl-AMP using ATP as a substrate) but prior to its transfer to the tRNA (pre-transfer editing). Proofreading can happen at a distinct editing site after the activated non-cognate amino acid has been esterified with the tRNA
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