Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase

Abstract

D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD’s invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD’s chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2′-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD’s activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting.

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

“…The substrate is, however, completely deacylated by 100 nM EcDTD even in the presence of EF-Tu. Thus, the protection offered by EF-Tu to Gly-tRNA Gly against EcDTD is about 10-fold (Figure 3a), which is in agreement with our previous study (Routh et al, 2016).…”

“…Our previous work had shown that elongation factor thermo unstable (EF-Tu) confers protection on Gly-tRNA Gly from DTD’s unwarranted activity (Routh et al, 2016). tRNA elements on TΨC arm are known to be responsible for EF-Tu binding (LaRiviere et al, 2001; Sanderson and Uhlenbeck, 2007; Schrader et al, 2009).…”

“…the enhancement in protection of Gly-tRNA Gly by EF-Tu is consequential only if the substrate harbors U73. Nevertheless, cellular DTD levels must be tightly regulated because DTD overexpression has been shown to cause toxicity (Routh et al, 2016). While the above data indicated the role of DTD in depleting the cognate Gly-tRNA Gly pool, it did not provide direct evidence to that effect.…”

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

“…Nevertheless, the glycine ‘misediting paradox’ was effectively resolved by safeguarding the cognate achiral substrate using EF-Tu as well as keeping the cellular levels of DTD low and tightly regulated (Routh et al, 2016). Thus, what seemed to be an apparent flaw in the architecture of DTD’s active site proved to be a necessity.…”

A chiral selectivity relaxed paralog of DTD for proofreading tRNA mischarging in Animalia

“…2H). By contrast, E. coli DTD (EcDTD) or PfDTD does not act on L-chiral substrates even at 100-fold higher concentration than required for D-chiral substrate (11,12). Therefore, biochemical probing suggested that ATD is an aminoacyl-tRNA deacylase with a relaxed specificity for substrate chirality, primarily due to the trans conformation of its active-site Gly-Pro motif.…”