Role of D-aminoacyl-tRNA deacylase beyond chiral proofreading as a cellular defense against glycine mischarging by AlaRS
- CSIR–Centre for Cellular and Molecular Biology, India
Figures
Figure 1
Mischarging by AlaRS.
AlaRS activates and charges alanine (A) to form cognate Ala-tRNAAla which is routed for protein synthesis. In this process, AlaRS also misactivates glycine (G) and serine (S) at frequencies of 1 per …
Figure 2 with 1 supplement
Misacylation of tRNAAla with glycine by AlaRS and its prevention/rectification by DTD.
(a) Aminoacylation of tRNAAla by EcAlaRS in the presence of activated EF-Tu: L-alanine (green square), L-alanine and 10 pM EcDTD (green triangle), glycine (pink square), glycine and 10 pM EcDTD …
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Figure 2—source data 1
Misacylation of tRNAAla and deacylation of Gly-tRNAAla in the presence of EF-Tu.
- https://doi.org/10.7554/eLife.24001.005
Figure 2—figure supplement 1
Accumulation of Ala/Gly/Ser-tRNAAla during aminoacylation by EcAlaRS C666A in the presence of EF-Tu.
Aminoacylation of tRNAAla by EcAlaRS C666A in the presence of activated EF-Tu: L-alanine (green square), glycine (pink square), L-serine (purple square).
Figure 3
DTD has higher activity than AlaRS for the editing of Gly-tRNAAla.
(a) Deacylation of Gly-tRNAAla in the presence of unactivated EF-Tu: buffer (blue diamond), 10 nM EcAlaRS (red circle), 50 nM EcAlaRS (green circle), 100 nM EcAlaRS (purple circle), 500 nM EcAlaRS …
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Figure 3—source data 1
Deacylation of Gly-tRNAAla in the presence of unactivated and activated EF-Tu.
- https://doi.org/10.7554/eLife.24001.008
Figure 4
E. coli AlaRS editing site mutants.
Homology model of E. coli AlaRS depicting serine (green sticks/spheres) in the editing site. E. coli AlaRS cis-editing domain was modeled using A. fulgidus AlaRS (PDB id: 2ZTG) as a template, …
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Figure 4—source data 1
Deacylation of Ser-tRNAAla and Gly-tRNAAla by E. coli AlaRS editing site mutants.
- https://doi.org/10.7554/eLife.24001.010
Figure 5 with 2 supplements
DTD knockout causes pronounced glycine toxicity in E. coli.
Spot dilution assay of E. coli MG1655 ∆alaS/para: : alaS-T567F, S587W, C666F compared with that of E. coli MG1655 ∆dtd, ∆alaS/para:: alaS-T567F, S587W, C666F (a) in the presence of no amino acid, 3 …
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Figure 5—source data 1
Growth curves of E. coli MG1655 with and without dtd knockout in AlaRS editing-defective background.
- https://doi.org/10.7554/eLife.24001.012
Figure 5—figure supplement 1
DTD is inactive on Ser-tRNAAla.
Deacylation of Ser-tRNAAla by buffer (blue diamond), 50 nM EcDTD (pink circle), 500 nM EcDTD (purple circle), 5 µM EcDTD (orange circle).
Figure 5—figure supplement 2
Spot dilution assay of E. coli MG1655 compared with E. coli MG1655 ∆dtd with increasing concentration of glycine.
https://doi.org/10.7554/eLife.24001.014
Figure 6 with 1 supplement
DTD positively selects the tRNA acceptor stem element G3•U70.
(a) Deacylation of Gly-tRNAGly by buffer (blue diamond), 5 nM EcDTD (purple square), 50 nM EcDTD (pink circle). (b) Deacylation of Gly-tRNAGly A3•U70 by buffer (blue diamond), 5 nM EcDTD (purple …
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Figure 6—source data 1
Deacylation of Gly-tRNAGly mutants and Gly-tRNAAla by DTD.
- https://doi.org/10.7554/eLife.24001.016
Figure 6—figure supplement 1
DTD’s activity on the cognate achiral substrate.
Deacylation of Gly-tRNAGly by buffer (blue diamond), 5 nM EcDTD (brown diamond). Error bars indicate one standard deviation from the mean of triplicate readings.
Figure 7 with 1 supplement
DTD edits Gly-tRNAAla across bacteria and eukaryotes.
(a) Deacylation of E. coli Gly-tRNAAla by buffer (blue diamond), 10 pM EcDTD (red square), 10 pM PfDTD (green triangle), 10 pM LmDTD (purple cross), 10 pM DmDTD (cyan star), 10 pM DrDTD (orange …
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Figure 7—source data 1
Deacylation of E. coli Gly-tRNAAla and D. melanogaster Gly-tRNAAla by bacterial and eukaryotic DTDs.
- https://doi.org/10.7554/eLife.24001.019
Figure 7—figure supplement 1
Variations in the tRNA-binding site of DTD.
(a) Structure-based multiple sequence alignment of DTD from different organisms. The residues which are within a distance of 6 Å from the 3′-terminal CCA-arm of tRNA are marked by green stars, and …
Figure 8
DTD doubles as a key factor to uncouple glycine mischarged on tRNAAla.
In the cell, aminoacylation by aaRSs leads to the formation of different aa-tRNAs, of which L-aa-tRNAs (left extreme) are not acted upon by DTD, while D-aa-tRNAs (right extreme) are effectively …
Author response image 1
E. coli tRNAGly isoacceptors showing identical acceptor stem.
https://doi.org/10.7554/eLife.24001.023Tables
Author response table 1
Protein concentration estimation.
| Protein Name | Concentration of protein taken (mg/ml) measured using A280 | Protein concentration estimation (mg/ml) using | |
|---|---|---|---|
| Bicinchoninic acid Assay | Bradford Assay | ||
| E. coli AlaRS | 4 | 3.82 | 5.22 |
| 8 | 7.45 | 9.02 | |
| E. coli DTD | 4 | 4.36 | 4.83 |
| 8 | 8.08 | 7.93 | |
