Fig. 5: Implanting ThrRS-Ed determinant in AlaRS-Ed affects its Zn2+ binding and ROS resistance of activity.
From: A metal ion mediated functional dichotomy encodes plasticity during translation quality control
a Schematic diagram showing PAR-based competitive assay for assessing Zn2+ binding strength. b Graph showing absorbance spectra across λ = 450 nm to 550 nm of PAR assay samples: 50 µM EcAlaRS WT with GdnCl (red), 50 µM EcAlaRS WT without GdnCl (green), 50 µM EcAlaRS L656Y with GdnCl (black) and 50 µM EcAlaRS L656Y without GdnCl (purple); (N = 3). c Graph showing absorbance spectra across λ = 450 nm to 550 nm of PAR assay samples: 50 µM Zinc acetate (blue), 50 µM EcN1 + N2 WT with GdnCl (red), 50 µM Ec N1 + N2 WT with GdnCl (green); (N = 3). d Deacylation of Ser-(Ec)tRNAAla by buffer (black square), 50 nM EcAlaRS WT (black triangle), 50 nM EcAlaRS WT + 5 mM H2O2 (black circle), 50 nM EcAlaRS WT + 10 mM H2O2 (black diamond); (N = 3). e Deacylation of Ser-(Ec)tRNAAla by buffer (black square), 50 nM EcAlaRS L656Y (black triangle), 50 nM EcAlaRS L656Y + 5 mM H2O2 (black circle), 50 nM EcAlaRS L656Y + 10 mM H2O2 (black diamond); (N = 3). f Bar plot showing level of sulfhydryl group in PhoAlaX-M T172Y upon treatment with H2O2 using Ellman’s reagent; (N = 3). g Spot dilution-based growth assay of E. coli MG1655 Δdtd ΔalaS strains complemented with either alaSWT or alaSL656Y in M9 minimal agar plate, M9 minimal agar plates supplemented with: 3 mM Ser, 3 mM Ser + 50 µM H2O2, 3 mM Ser + 50 µM H2O2 + 10 mM Ala. Representative plates of three independent experiments are shown. In all the graphs in the panel, data are represented as mean ± standard deviation. Statistical analysis in (f) was done using unpaired two-sided Student’s t test. Source data are provided in the Source Data file.
