Fig. 3: Zn2+ protects AlaRS editing activity from ROS in vivo.
From: A metal ion mediated functional dichotomy encodes plasticity during translation quality control
a Schematic diagram showing HXXXH and CXGXH Zn2+ binding motifs, residues marked with symbol ‘#’ indicates residues whose mutation affects activity and symbol ‘*’ represent residues with minor consequence on activity upon mutation. b Deacylation of Ser-(Ec)tRNAAla by buffer (black diamond), 50 nM EcAlaRSH568A (purple diamond), 50 nM EcAlaRSH568A + 50 µM H2O2 (green diamond), 50 nM EcAlaRSH568A + 100 µM H2O2 (blue diamond), 50 nM EcAlaRSH568A + 1 mM H2O2 (pink diamond), 50 nM EcAlaRSH568A + 5 mM H2O2 (orange diamond); (N = 3). c Graph showing deacylation of Ala-(Ec)tRNAAla by buffer (black diamond), 50 nM EcAlaRSH568A (black triangle); (N = 3). d Bar plot showing level of sulfhydryl group in PhoAlaX-M H103A using Ellman’s reagent; (N = 3). e Spot dilution-based growth assay of E. coli MG1655 Δdtd ΔalaS strains complemented with either alaSWT or alaSH568A in M9 minimal agar plate under conditions: control, 3 mM Ser, 3 mM Ser + 50 µM H2O2, 3 mM Ser + 50 µM H2O2 + 10 mM Ala, 10 mM Gly, 10 mM Gly + 50 µM H2O2, 10 mM Gly + 50 µM H2O2 + 10 mM Ala. Representative plates of three independent experiments are shown. f A GFP-based cellular reporter of Ala-to-Gly mistranslation, where GFP G67A mutant having 65TYA50 non-fluorescent chromophore tripeptide regains fluorescence upon translation of GFP with G67 as a consequence of the cellular Ala-to-Gly mistranslation, thereby reports Ala-codon mistranslation in a cell as a function of GFP fluorescence (Kuncha et al.49). g Fluorescence microscopy images showing GFP fluorescence of E. coli MG1655 Δdtd ΔalaS Para::gfpG67A strains complemented with either alaSWT or alaSH568A grown in control and in presence of 10 mM glycine + 50 µM H2O2. Representative images of three independent experiments are shown and scale bar = 4.5 µm. In all the graphs in the panel, data are represented as mean ± standard deviation. Statistical analysis in (d) was done using unpaired two-sided Student’s t test. Source data are provided in the Source Data file.
