A translation proofreader of archaeal origin imparts multi-aldehyde stress tolerance to land plants.

Aldehydes, being an integral part of carbon metabolism, energy generation, and signalling pathways, are ingrained in plant physiology. Land plants have developed intricate metabolic pathways which involve production of reactive aldehydes and its detoxification to survive harsh terrestrial environments. Here, we show that physiologically produced aldehydes, i.

Distinct localization of chiral proofreaders resolves organellar translation conflict in plants.

Plants have two endosymbiotic organelles originated from two bacterial ancestors. The transition from an independent bacterium to a successful organelle would have required extensive rewiring of biochemical networks for its integration with archaeal host. Here, using as a model system, we show that plant D-aminoacyl-tRNA deacylase 1 (DTD1), of bacterial origin, is detrimental to organellar protein synthesis owing to its changed tRNA recognition code.

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

Mitochondria emerged through an endosymbiotic event involving a proteobacterium and an archaeal host. However, the process of optimization of cellular processes required for the successful evolution and survival of mitochondria, which integrates components from two evolutionarily distinct ancestors as well as novel eukaryotic elements, is not well understood. We identify two key switches in the translational machinery—one in the discriminator recognition code of a chiral proofreader DTD [d-aminoacyl–transfer RNA (tRNA) deacylase] and the other in mitochondrial tRNA—that enable the compatibility between disparate elements essential for survival.

Recruitment of archaeal DTD is a key event toward the emergence of land plants.

Streptophyte algae emerged as a land plant with adaptations that eventually led to terrestrialization. Land plants encounter a range of biotic and abiotic stresses that elicit anaerobic stress responses. Here, we show that acetaldehyde, a toxic metabolite of anaerobic stress, targets and generates ethyl adducts on aminoacyl-tRNA, a central component of the translation machinery.

Genomic innovation of ATD alleviates mistranslation associated with multicellularity in Animalia.

The emergence of multicellularity in Animalia is associated with increase in ROS and expansion of tRNA-isodecoders. tRNA expansion leads to misselection resulting in a critical error of L-Ala mischarged onto tRNA, which is proofread by Animalia-specific-tRNA Deacylase (ATD) in vitro. Here we show that in addition to ATD, threonyl-tRNA synthetase (ThrRS) can clear the error in cellular scenario.

Chiral checkpoints during protein biosynthesis.

Protein chains contain only l-amino acids, with the exception of the achiral glycine, making the chains homochiral. This homochirality is a prerequisite for proper protein folding and, hence, normal cellular function. The importance of d-amino acids as a component of the bacterial cell wall and their roles in neurotransmission in higher eukaryotes are well-established.

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

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-tRNA with 1000-fold higher efficiency than its activity on Gly-tRNA, indicating tRNA-based modulation of DTD (Pawar et al., 2017).

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

Strict L-chiral rejection through Gly-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 '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 species four orders of magnitude more efficiently than even AlaRS, the only ubiquitous cellular checkpoint known for clearing the error.

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

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.

Specificity and catalysis hardwired at the RNA-protein interface in a translational proofreading enzyme.

Proofreading modules of aminoacyl-tRNA synthetases are responsible for enforcing a high fidelity during translation of the genetic code. They use strategically positioned side chains for specifically targeting incorrect aminoacyl-tRNAs. Here, we show that a unique proofreading module possessing a D-aminoacyl-tRNA deacylase fold does not use side chains for imparting specificity or for catalysis, the two hallmark activities of enzymes.

Mechanism of chiral proofreading during translation of the genetic code.

The biological macromolecular world is homochiral and effective enforcement and perpetuation of this homochirality is essential for cell survival. In this study, we present the mechanistic basis of a configuration-specific enzyme that selectively removes D-amino acids erroneously coupled to tRNAs. The crystal structure of dimeric D-aminoacyl-tRNA deacylase (DTD) from Plasmodium falciparum in complex with a substrate-mimicking analog shows how it uses an invariant 'cross-subunit' Gly-cisPro dipeptide to capture the chiral centre of incoming D-aminoacyl-tRNA.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analyses of threonyl-tRNA synthetase editing domain from Aeropyrum pernix.

The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeon Pyrococcus abysii in complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms.

Mechanistic insights into cognate substrate discrimination during proofreading in translation.

Editing/proofreading by aminoacyl-tRNA synthetases is an important quality control step in the accurate translation of the genetic code that removes noncognate amino acids attached to tRNA. Defects in the process of editing result in disease conditions including neurodegeneration. While proofreading, the cognate amino acids larger by a methyl group are generally thought to be sterically rejected by the editing modules as envisaged by the "Double-Sieve Model.

Post-transfer editing mechanism of a D-aminoacyl-tRNA deacylase-like domain in threonyl-tRNA synthetase from archaea.

To ensure a high fidelity during translation, threonyl-tRNA synthetases (ThrRSs) harbor an editing domain that removes noncognate L-serine attached to tRNAThr. Most archaeal ThrRSs possess a unique editing domain structurally similar to D-aminoacyl-tRNA deacylases (DTDs) found in eubacteria and eukaryotes that specifically removes D-amino acids attached to tRNA. Here, we provide mechanistic insights into the removal of noncognate L-serine from tRNAThr by a DTD-like editing module from Pyrococcus abyssi ThrRS (Pab-NTD).

A D-amino acid editing module coupled to the translational apparatus in archaea.

We report the crystal structure of an archaea-specific editing domain of threonyl-tRNA synthetase that reveals a marked structural similarity to D-amino acid deacylases found in eubacteria and eukaryotes. The domain can bind D-amino acids despite a low sequence identity to other D-amino acid deacylases. These results together indicate the presence of these deacylases in all three kingdoms of life.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic investigations of a unique editing domain from archaebacteria.

Threonyl-tRNA synthetase (ThrRS) faces a crucial double-discrimination problem during the translation of genetic code. Most ThrRSs from the archaeal kingdom possess a unique editing domain that differs from those of eubacteria and eukaryotes. In order to understand the structural basis of the editing mechanism in archaea, the editing module of ThrRS from Pyrococcus abyssi comprising of the first 183 amino-acid residues was cloned, expressed, purified and crystallized.