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

Abstract

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 non-cognate Gly-tRNAAla with 1000-fold higher efficiency than its activity on Gly-tRNAGly, 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-tRNAGly 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 tRNAGly/Ala discrimination. Moreover, DTD prevents alanine-to-glycine misincorporation in proteins rather than only recycling mischarged tRNAAla. Overall, the study reveals the unique co-evolution of DTD and discriminator base, and suggests DTD’s strong selection pressure on bacterial tRNAGlys to retain a pyrimidine discriminator code.

Switching a conflicted bacterial DTD-tRNA code is essential for the emergence of mitochondria

“…It was puzzling how DTD in eukaryotes avoids misediting of Gly-tRNA Gly , which harbors adenine at the 73rd position. We earlier noted a switch in eukaryotic DTD’s discriminator code preference using Mus musculus DTD and Escherichia coli tRNA Gly U73 (wild type, WT) and ( Ec )tRNA Gly U73A mutant ( 11 ); however, the universality of such a shift across eukaryotes and its physiological necessity in avoiding Gly-tRNA Gly misediting was unexplored.…”

“…A discriminator code was also elucidated for bacterial DTD according to which pyrimidine at the N73rd position in Gly-tRNAs acts as a negative determinant for DTD and thereby prevents Gly-tRNA Gly misediting, whereas Gly-tRNAs bearing purine at the N73rd position as in Gly-tRNA Ala is hydrolyzed robustly by DTD. DTD from bacteria has been shown to have 100-fold more activity on Gly-tRNA Gly bearing A73 than the ones having U73 ( 11 ).…”

The G3-U70-independent tRNA recognition by human mitochondrial alanyl-tRNA synthetase

“…However, Ser was excluded from misincorporation only at the post-transfer editing level. Besides post-transfer editing by AlaRS itself, AlaRS-produced Gly-tRNA Ala is further excluded by the additional checkpoint provided by the universal DTD in bacteria, archaeal and human cytoplasm (40,53). DTD recognizes tRNA Ala in a G3-U70-dependent manner (40).…”

Aminoacyl-tRNA synthetases

“…In addition to editing D-amino acids, DTDs have also been shown to be able to hydrolyze the achiral glycine and edit Gly-tRNA Ala (Pawar et al 2017) while preventing editing of the cognate Gly-tRNA Gly via a conserved discriminator base at position 73 (Kuncha et al 2018).…”