Mechanism of chiral proofreading during translation of the genetic code

Ahmad, Sadeem; Routh, Satya Brata; Kamarthapu, Venu; Chalissery, Jisha; Muthukumar, Sowndarya; Hussain, Tanweer; Kruparani, Shobha P; Deshmukh, Mandar V.; Sankaranarayanan, Rajan

doi:10.7554/elife.01519

Cited by 46 publications

(83 citation statements)

References 44 publications

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“…Both the Bacterial (Ec) and Eukaryotic (At) DTD1s did not act on NELATs and also on NEDATs (Fig: 2C, 2D) (Extended Data Fig: 5). DTD1's lack of activity on NEDATs is further corroborated by the earlier crystal structure of DTD1 in complex with substrate analog D-Tyr3AA 6,15 . Wherein the D-amino acid of D-aminoacyl-tRNA snugly fits into the active site of DTD1, thereby sterically excludes N-ethylated amino group of D-amino acid ( Fig: 2E).…”

Section: Dtd2 Alleviates Anaerobic Stress In Plants By Recycling Nedatssupporting

confidence: 54%

“…Apart from plants, DTD2 is also conserved in Archaea ( We next interrogated substrate specificity of DTD2 in terms of modification by performing deacylations with N-acetyl-D-aa-tRNAs and found that both, archaeal (Pho) and plant (At) DTD2s did not hydrolyse acetyl substrates (Fig: 3G, H) suggesting, DTD2 is specific for ethyl modification and evolved exclusively for ND activity. It is therefore intriguing to probe 6 what physiological constraints necessitated the conservation of DTD2 and its ND activity in Archaea and plants. To this end, we searched for common physiological and biochemical characteristics among Archaea and plants.…”

Section: Nd Activity Is Rooted In the Archaeamentioning

confidence: 99%

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Recruitment of Archaeal DTD is a Key Event in the Emergence of Land Plants

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Singh

Kumar

et al. 2020

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Land plant evolution is a major leap in the history of life that took place during the Neoproterozoic Era (~800 Mya). Charophyceae, a class of rhyzophytic green algae emerged as a land plant with innovations in biochemical, cytological and developmental adaptations and played a crucial role in establishing life on the land 1,2 . One such striking architectural innovation is "root" that experience harsh environmental assaults such as floods, waterlogging and therefore is the epicentre for anaerobic fermentation, which produces toxic acetaldehyde 3 . Here, we show that such produced acetaldehyde makes N-ethyl-adducts on a central component of translation machinery aa-tRNA. The Plant kingdom is unique among life forms in possessing two chirality-based proofreading systems represented by Daminoacyl-tRNA deacylases (DTD1 and DTD2), derived from Bacteria and Archaea 4 . We identified a unique role of archaeal derived chiral proofreading module DTD2 that selectively deacylates N-ethyl-D-aminoacyl-tRNAs (NEDATs) in plants. NEDAT deacylase function is exclusive to DTD2, as no other proofreading modules with similar substrates like canonical DTD1 and peptidyl-tRNA hydrolase (PTH) can clear NEDATs. Thus, the study elucidates the cause of hypersensitivity of DTD2 knockout plants for both ethanol and acetaldehyde. We further show NEDAT elimination is rooted in Archaea which possess the biosynthesis machinery for ethanol fermentation similar to plants. While absent in other algal branches, DTD2 can be identified in plants from land plant ancestors-Charophytes onwards. DTD2 is the only gene that has only archaeal origin among the genes ascribed for architectural and genomic innovations that happened in the land plant ancestors. The work has uncovered an important gene transfer event from methanogenic archaea to the charophytes in the oldest terrestrial ecosystem bog that contains excess of D-amino acids and deprived of oxygen. 3 Plants being sedentary in their lifestyle are dependent on roots for their nourishment and root has to survive in the oxygen-deprived environment of soil. The soil also being enriched with D-amino acids makes roots clutched with inescapable stress of D-amino acids compounded with anaerobic stress. The presence of D-amino acids in plants is inevitable and play vital roles in plant physiology such as the development of the pollen tube and also act as a major nitrogen source 5 . On the contrary, D-amino acids also cause cellular toxicity by generating Daminoacyl-tRNAs (D-aa-tRNAs) which arrests free tRNA pool from translation. Removal of mischarged D-amino acids from D-aa-tRNAs is termed as chiral proofreading 6 that enables the perpetuation of homochirality in the cellular proteome 7 . D-amino acid editing modules are universally conserved; D-aminoacyl-tRNA deacylase 1 (DTD1) is present in both Bacteria and Eukaryotes and DTD2 is in Archaea and plants 4 . Plants are the only organisms that possess both the evolutionarily distinct chiral proofreading modules DTD1 and DTD2 8,9 . Surprisingly, DTD2 knock out...

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Section: Dtd2 Alleviates Anaerobic Stress In Plants By Recycling Nedatssupporting

confidence: 54%

Section: Nd Activity Is Rooted In the Archaeamentioning

confidence: 99%

Recruitment of Archaeal DTD is a Key Event in the Emergence of Land Plants

Mazeed

Singh

Kumar

et al. 2020

Preprint

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“…The other PDB entries with less sequence similarity or coverage, listed in Table S2, are oxidoreductases, (5ID2, 3DRN), deacylases (1J7G, 2DBO, 1JKE, 3KNP, 3KO3, 3KO4, 4NBJ, 4NBI, 5J61), a PH domain of Evectin‐2 (2DHI) and an oligomerization domain of Dmp53 (2RP4). The structural and sequential coverage of these proteins with a known structure and function is not high enough to make a reliable assumption of the possible cellular function of HVO_2922 (Figure ).…”

Section: Resultsmentioning

confidence: 99%

Solution Structure and Dynamics of the Small Protein HVO_2922 from Haloferax volcanii

et al. 2019

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Past sequencing campaigns overlooked small proteins as they seemed to be irrelevant due to their small size. However, their occurrence is widespread, and there is growing evidence that these small proteins are in fact functionally very important in organisms found in all kingdoms of life. Within a global proteome analysis for small proteins of the archaeal model organism Haloferax volcanii, the HVO_2922 protein has been identified. It is differentially expressed in response to changes in iron and salt concentrations, thus suggesting that its expression is stress‐regulated. The protein is conserved among Haloarchaea and contains an uncharacterized domain of unknown function (DUF1508, UPF0339 family protein). We elucidated the NMR solution structure, which shows that the isolated protein forms a symmetrical dimer. The dimerization is found to be concentration‐dependent and essential for protein stability and most likely for its functionality, as mutagenesis at the dimer interface leads to a decrease in stability and protein aggregation.

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“…The genes encoding E. coli DTD and Thermus thermophilus EF-Tu, and cDNA encoding M. musculus (residues 1-147) were cloned (with C-terminal 6X His-tag), expressed and purified as mentioned in Ahmad et al, 2013 and Kuncha et al, 2018. Briefly the proteins were overexpressed in E. coli BL21(DE3) by growing the culture to 0.6 OD (600 nM) at 37°C, followed by induction using 0.5 mM IPTG for 12 hours at 18°C.…”

Section: Methodsmentioning

confidence: 99%

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

Sankaranarayanan

Kuncha

Suma

et al. 2018

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10D-aminoacyl-tRNA deacylase (DTD) acts on achiral glycine, in addition to D-amino acids, 11 attached to tRNA. We have recently shown that this activity enables DTD to clear non-cognate 12 Gly-tRNA Ala with 1000-fold higher efficiency than its activity on Gly-tRNA Gly , indicating 13 tRNA-based modulation of DTD (Pawar et al., 2017). Here, we show that tRNA's 14 discriminator base predominantly accounts for this activity difference and is the key to 15 selection by DTD. Accordingly, the uracil discriminator base, serving as a negative 16 determinant, prevents Gly-tRNA Gly misediting by DTD and this protection is augmented by 17 EF-Tu. Intriguingly, eukaryotic DTD has inverted discriminator base specificity and uses only 18 G3•U70 for tRNA Gly/Ala discrimination. Moreover, DTD prevents alanine-to-glycine 19 misincorporation in proteins rather than only recycling mischarged tRNA Ala . Overall, the study 20 reveals the unique co-evolution of DTD and discriminator base, "reciprocally" in Bacteria and 21 Eukarya, and suggests DTD's strong selection pressure on bacterial tRNA Gly s to retain a 22 pyrimidine discriminator code. 23 24 Quality control during translation of the genetic code involves multiple stages and a multitude 25 of proofreading factors (Guo and Schimmel, 2012; Ibba and Söll, 2000; Ogle and 26 Ramakrishnan, 2005). A compromise in editing leads to serious pathologies including 27 neurodegeneration in mouse, and even cell death (Bacher et al., 2005; Bullwinkle et al., 2014; 28 Karkhanis et al., 2007; Korencic et al., 2004; Lee et al., 2006; Liu et al., 2014; Lu et al., 29 2014; Moghal et al., 2016; Nangle et al., 2002; Roy et al., 2004). Among these proofreading 30 factors, D-aminoacyl-tRNA deacylase (DTD) is the one that specifically decouples wrongly 31 acylated D-amino acids from tRNAs (Calendar and Berg, 1967; Soutourina et al., 1999, 32 2000). Our studies have shown that DTD is an RNA-based catalyst that uses an invariant Gly-33 cisPro motif as a "chiral selectivity filter" to achieve substrate chiral specificity only through 34 rejection of L-amino acid from the active site, thereby leading to Gly-tRNA Gly misediting 35 (Ahmad et al., 2013; Routh et al., 2016; Routh and Sankaranarayanan, 2017). Recently, 36we have also shown that DTD's activity on achiral glycine helps in clearing Gly-tRNA Ala , a 37 misaminoacylation product of alanyl-tRNA synthetase (AlaRS), thus resolving a long-standing 38 question in translational quality control (Pawar et al., 2017). 39 As DTD acts on multiple tRNAs charged with D-amino acids or glycine, tRNA's role 40 in modulating DTD's activity was previously thought to be inconsequential (Calendar and 41 Berg, 1967). However, our recent work has shown that DTD's activity on Gly-tRNA Ala is about 42 1000-fold higher than on Gly-tRNA Gly , clearly demonstrating the profound effect of tRNA 43 elements on DTD and suggesting an underlying tRNA code for DTD's action. G3•U70, the 44 universal tRNA Ala -specific determinant for AlaRS, is also a determinant for DTD. Ho...

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Mechanism of chiral proofreading during translation of the genetic code

Cited by 46 publications

References 44 publications

Recruitment of Archaeal DTD is a Key Event in the Emergence of Land Plants

Recruitment of Archaeal DTD is a Key Event in the Emergence of Land Plants

Solution Structure and Dynamics of the Small Protein HVO_2922 from Haloferax volcanii

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

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