Satya Brata Routh

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. While no protein residues are directly involved in catalysis, the unique side chain-independent mode of substrate recognition provides a clear explanation for DTD’s ability to act on multiple D-amino acids. The strict chiral specificity elegantly explains how the enriched cellular pool of L-aminoacyl-tRNAs escapes this proofreading step. The study thus provides insights into a fundamental enantioselection process and elucidates a chiral enforcement mechanism with a crucial role in preventing D-amino acid infiltration during the evolution of translational apparatus. DOI: http://dx.doi.org/10.7554/eLife.01519.001

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. We show, using three distinct archaea, that a side-chain-stripped recognition site is fully capable of solving a subtle discrimination problem. While biochemical probing establishes that RNA plays the catalytic role, mechanistic insights from multiple high-resolution snapshots reveal that differential remodelling of the catalytic core at the RNA–peptide interface provides the determinants for correct proofreading activity. The functional crosstalk between RNA and protein elucidated here suggests how primordial enzyme functions could have emerged on RNA–peptide scaffolds before recruitment of specific side chains.

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. 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.

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

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. DOI: http://dx.doi.org/10.7554/eLife.24001.001

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

D-aminoacyl-tRNA deacylase (DTD), a bacterial/eukaryotic trans-editing factor, removes d-amino acids mischarged on tRNAs and achiral glycine mischarged on tRNAAla. An invariant cross-subunit Gly-cisPro motif forms the mechanistic basis of l-amino acid rejection from the catalytic site. Here, we present the identification of a DTD variant, named ATD (Animalia-specific tRNA deacylase), that harbors a Gly-transPro motif. The cis-to-trans switch causes a “gain of function” through L-chiral selectivity in ATD resulting in the clearing of l-alanine mischarged on tRNAThr(G4•U69) by eukaryotic AlaRS. The proofreading activity of ATD is conserved across diverse classes of phylum Chordata. Animalia genomes enriched in tRNAThr(G4•U69) genes are in strict association with the presence of ATD, underlining the mandatory requirement of a dedicated factor to proofread tRNA misaminoacylation. The study highlights the emergence of ATD during genome expansion as a key event associated with the evolution of Animalia.The number of tRNA isodecoders has expanded significantly during evolution, which has resulted in ambiguity in tRNA selection by synthetases. Here the authors identify and characterize a dedicated proofreading factor that eliminates errors caused by ambiguity in tRNA selection by eukaryotic tRNA synthetases.

Functions of D-aminoacyl-tRNA deacylase in Drosophila melanogaster

of the pituitary gland, a critical organ that regulates vital physiological processes in vertebrates. Despite the importance of the RAS-RAF-MAPK pathway in normal physiology and disease of numerous organs, its role during pituitary development and tumourigenesis remains largely unknown. Here we use a genetic approach to elevate MAPK signalling by conditionally expressing the gain-of-function alleles BrafV600E and KrasG12D in the developing mouse pituitary. We show that the overactivation of theMAPK pathway during embryonic development results in severe hyperplasia and abnormal morphogenesis of the pituitary gland by the end of gestation. Cell-lineage commitment and terminal differentiation are disrupted leading to a significant reduction in numbers of most of the hormone-producing cells before birth, with the exception of corticotrophs. Of note, Sox2+ve stem cells and clonogenic potential are drastically increased in the mutant pituitaries. Finally, we reveal that papillary craniopharyngioma (PCP), a benign human pituitary tumour harbouring BRAF p.V600E contains Sox2+ve cells with sustained proliferative capacity and disrupted pituitary differentiation. Together, our data demonstrate a critical function of theMAPKpathway in controlling the balance between proliferation and differentiation of Sox2+ve cells and suggest that persistent proliferative capacity of Sox2+ve cells may underlie the pathogenesis of PCP.

Mechanistic insights into chiral proofreading during translation of genetic code

Exclusion of D-amino acids from the translational apparatus is essential for protein homeostasis and cellular integrity. An enzyme named D-aminoacyl-tRNA deacylase (DTD) decouples D-amino acids that have been mischarged on tRNAs, thereby preventing cellular toxicity due to D-amino acids [1]. DTD has thus been implicated as a key cellular chiral checkpoint during protein biosynthesis. Using ligand-bound crystal structures in conjunction with mutational and biochemical analyses, we have deciphered the mechanisms of enantioselectivity and catalysis of this chiral proofreading enzyme. DTD is extremely enantioselective because it does not act on even the smallest amino acid with L-chirality, i.e., L-alanine [2,3]. It employs an invariant, cross-subunit Gly-cisPro motif to ensure stereospecificity during substrate selection. The rigid fixation of the cis conformation of the motif disposes the two carbonyl oxygens in a near parallel orientation, which forms the structural basis of DTD’s enantioselectivity. Any perturbation of the motif renders the enzyme inactive, thereby suggesting its importance in the maintenance of the active site architecture [2]. However, the enzyme functions only through strict L-chiral rejection and not Dchiral selection. DTD’s chiral proofreading site is therefore porous to achiral glycine which in turn leads to glycine “misediting paradox”. Nevertheless, elongation factor thermo unstable (EF-Tu) protects the achiral substrate Gly-tRNAGly against DTD’s unwarranted activity [3]. On the basis of ligand-bound crystal structures of a structural homolog of DTD, we had proposed that DTD uses the 2′-OH of A76 of tRNA for catalysis. Our mutational studies later demonstrated that active site residues of DTD are dispensable for catalysis [2]. Using 2′-modified tRNAs, we have recently shown that DTD employs the 2′-OH to effect hydrolysis of its substrates [2]. Thus, DTD is a primordial RNA-based enzyme which functions at the RNA–protein interface.

D-Chiral specificity of archaeal and cyanobacterial D-aminoacyl-tRNA deacylases

Biological macromolecular world is homochiral. D-aminoacyl-tRNA deacylase (DTD) plays an important role in maintaining homochirality of proteins by removing mistakenly attached D-amino acids from tRNAs [1]. In nature, three distinct DTDs exist, namely DTD1, DTD2 and DTD3. DTD1 is present in bacteria and eukaryotes, but absent in most archaea and cyanobacteria. DTD function is carried out by DTD2 in archaea and by DTD3 in cyanobacteria. Additionally, DTD2 is also reported in several plants. Interestingly, DTD1, DTD2 and DTD3 perform similar function without sharing any sequence similarity and structural homology among each other. Surprisingly, DTD2 shares structural similarity with a peptidyl-tRNA hydrolase, which is an essential enzyme. We have solved the first crystal structure of DTD3 at 1.68 Å, which is a structural homolog of DNase TatD. Strikingly, the positions of active site elements are also conserved between DTD3 and TatD. Active site of DTD1 has a fundamental design flaw by being porous to the smallest and achiral amino acid glycine, making it incapable of discriminating between glycine and D-amino acids, leads to glycine “misediting paradox” [2,3]. Biochemical analysis of DTD2 and DTD3 revealed absolute D-chiral specificity, which does not cross-react with even the smallest and achiral glycine. Attempts are underway to understand this mechanism of absolute D-chiral configuration specificity of DTD2 and DTD3. Remarkably, the role of 2′-OH of A76 of tRNA is indispensable for catalysis in all the three DTDs.

Editing and Proofreading in Translation

Translation of the genetic code is a complex and highly coordinated process orchestrated by many factors. Every step during the process undergoes strict quality control to ensure that errors are kept within tolerable limits. Besides aminoacyl-tRNA synthetases and ribosome, many other molecules and pathways contribute significantly to the overall fidelity of protein biosynthesis and, consequently, to cellular homeostasis. However, under adverse circumstances, errors can preferentially allow the cell/organism to adapt and evolve. Thus, editing/proofreading during translation is crucial, but can be modulated to help the organism to survive.