Abstract

Strict L-chiral rejection through Gly-cisPro motif during chiral proofreading underlies 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.

Article and author information

Author details

  1. Komal Ishwar Pawar

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
  2. Katta Suma

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
  3. Ayshwarya Seenivasan

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
  4. Santosh Kumar Kuncha

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
  5. Satya Brata Routh

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
  6. Shobha P Kruparani

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    Competing interests
    The authors declare that no competing interests exist.
  7. Rajan Sankaranarayanan

    CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
    For correspondence
    sankar@ccmb.res.in
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4524-9953

Funding

Council of Scientific and Industrial Research (12th Five Year Plan Project BSC0113)

  • Rajan Sankaranarayanan

Science and Engineering Research Board (JC Bose Fellowship)

  • Rajan Sankaranarayanan

Department of Biotechnology , Ministry of Science and Technology (Centre of Excellence)

  • Rajan Sankaranarayanan

Council of Scientific and Industrial Research (Research Fellowship)

  • Komal Ishwar Pawar
  • Satya Brata Routh

Department of Biotechnology , Ministry of Science and Technology (Research Associateship)

  • Katta Suma

Department of Biotechnology , Ministry of Science and Technology (INSPIRE Fellowship)

  • Santosh Kumar Kuncha

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2017, Pawar et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,487
    views
  • 395
    downloads
  • 33
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Komal Ishwar Pawar
  2. Katta Suma
  3. Ayshwarya Seenivasan
  4. Santosh Kumar Kuncha
  5. Satya Brata Routh
  6. Shobha P Kruparani
  7. Rajan Sankaranarayanan
(2017)
Role of D-aminoacyl-tRNA deacylase beyond chiral proofreading as a cellular defense against glycine mischarging by AlaRS
eLife 6:e24001.
https://doi.org/10.7554/eLife.24001

Share this article

https://doi.org/10.7554/eLife.24001

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Yamato Niitani, Kohei Matsuzaki ... Michio Tomishige
    Research Article

    The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for head–head coordination; however, which kinetic step(s) in the chemomechanical cycle is ‘gated’ by the neck linker remains unresolved. Here, we employed pre-steady-state kinetics and single-molecule assays to investigate how the neck-linker conformation affects kinesin’s motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubule, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.

    1. Biochemistry and Chemical Biology
    2. Computational and Systems Biology
    A Sofia F Oliveira, Fiona L Kearns ... Adrian J Mulholland
    Short Report

    The spike protein is essential to the SARS-CoV-2 virus life cycle, facilitating virus entry and mediating viral-host membrane fusion. The spike contains a fatty acid (FA) binding site between every two neighbouring receptor-binding domains. This site is coupled to key regions in the protein, but the impact of glycans on these allosteric effects has not been investigated. Using dynamical nonequilibrium molecular dynamics (D-NEMD) simulations, we explore the allosteric effects of the FA site in the fully glycosylated spike of the SARS-CoV-2 ancestral variant. Our results identify the allosteric networks connecting the FA site to functionally important regions in the protein, including the receptor-binding motif, an antigenic supersite in the N-terminal domain, the fusion peptide region, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected link between different allosteric sites. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spike revealed that glycans do not qualitatively change the internal allosteric pathways but can facilitate the transmission of the structural changes within and between subunits.