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
Ubiquitin activity-based
probes have proven invaluable in elucidating
structural mechanisms in the ubiquitin system by stabilizing transient
macromolecular complexes of deubiquitinases, ubiquitin-activating
enzymes, and the assemblies of ubiquitin-conjugating enzymes with
ubiquitin ligases of the RING-Between-RING and RING-Cysteine-Relay
families. Here, we demonstrate that an activity-based probe, ubiquitin-propargylamine,
allows for the preparative reconstitution and structural analysis
of the interactions between ubiquitin and certain HECT ligases. We
present a crystal structure of the ubiquitin-linked HECT domain of
HUWE1 that defines a catalytically critical conformation of the C-terminal
tail of the ligase for the transfer of ubiquitin to an acceptor protein.
Moreover, we observe that ubiquitin-propargylamine displays selectivity
among HECT domains, thus corroborating the notion that activity-based
probes may provide entry points for the development of specific, active
site-directed inhibitors and reporters of HECT ligase activities.
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