Figures and data
Suppressor screen identifies gidB as a potential fidelity factor.
A. Indirect aminoacylation pathway for tRNAAsn and tRNAGln in mycobacteria. B. Design of the suppressor screen: applying an increasing mistranslation stress to both strains makes HWS.19 more susceptible to stress and more likely to have mutation in suppressors. C. Workflow of suppressor screen. D. N to D mistranslation rate of suppressor candidates compared with WT and HWS.19 measured by Renilla-Firefly dual luciferase reporter as depicted in Figure1C. E. gidB mutation was mapped onto 3 candidates by whole genome sequencing. (*P < 0.05, **P < 0.01, ***P < 0.001, Student t test)
Deletion of GidB in mycobacteria increases translation fidelity in high mistranslation mutant.
A. N to D Mistranslation rate measured by gain-of-function Renilla-Firefly dual luciferase reporter in high mistranslation HWS19 strain (Left) and wild-type strain (Right). B. Q to E Mistranslation rate measured by gain-of-function dual fluorescent reporter in high mistranslation HWS19 strain (Left) and wild-type strain (Right).
Deletion of GidB in mycobacteria increases translation fidelity under high mistranslation physiological context.
A. Schematic of measuring mistranslation rate using gain-of-function dual fluorescence reporter under normal and high mistranslation physiological context B. Q to E mistranslation of mycobacteria scraped from LB-agar or LB-agar with low dose rifampicin. C. Gating strategy to gate top/bottom 10% of bacteria with high/low mistranslation rate. D. Q to E mistranslation of different bacteria population (no selection) gated from the highest/lowest mistranslation rate as described in C. E. Q to E mistranslation of different bacteria population (Rif selection) gated from the highest/lowest mistranslation rate as described in C (*P < 0.05, **P < 0.01, ***P < 0.001, Student t test)
Deletion of GidB decreases mistranslation mediated by non-physiological ‘misacylated-tRNA’.
A. The anticodon of an alanine-tRNA is mutated to tryptophan codon (EMAW) result in mistranslation from tryptophan to alanine (Right) to mimic non-physiological miacylated-tRNA compared to the two natural misacylated-tRNAs (Left) in mycobacteria. Mistranslation rate of tryptophan to alanine measured by Renilla-Firefly reporter (top) in high mistranslation HWS19 strain (B) and wild-type strain (C) with or without EMAW expression. (*P < 0.05, **P < 0.01, ***P < 0.001, Student t test)
Deletion of GidB decreases rifampicin tolerance.
Rifampicin killing curve in high mistranslation background (A) and wild-type background (B). T.test was performed between gidB deletion strain and the other two strains respectively. (*P < 0.05, **P < 0.01, ***P < 0.001, Student t test)
Structures of Mycobacterial Ribosomes with and without GidB-mediated methylation.
Ribosomal structures from the following Mycobacterium smegmatis stains: wild type, ΔgidB, HWS-19 and HWS-19-ΔgidB strains. A) wild-type: Different views of the Msm-70S structure (particle number = 591,747, 3.47 Å resolution) and B) zoomed in site of the GidB methyltransferase target residue (G507). C) ΔgidB: Different views of the Msm-70S structure (particle number = 1,466,88, resolution = 2.47 Å) and D) zoomed in site of the GidB methyltransferase target residue (G507). E) HWS-19: Different views of the Msm-70S structure (particle number = 108,034, resolution = 3.26 Å) and F) zoomed in site of the GidB methyltransferase target residue (G507) and interacting residues. G) HWS-19-ΔgidB: Different views of the Mycobacterium smegmatis 70S ribosomal structure (particle number = 136,418, map resolution = 3.07 Å) and H) zoomed in site of the GidB methyltransferase target residue (G507) and interacting residues.
Proposed model of gidB mediated translation fidelity control.
Under low mistranslation context (Left), wild-type ribosome and ΔGidB both have moderate translation fidelity. Under high mistranslation context (Right) due to impaired indirect aminoacylation pathway, HWS19 ribosome has a loosen ribosome conformation compared to HWS19-ΔGidB ribosome, which translation is error-prone. HWS19-ΔGidB ribosome restores structure integrity could better discriminate against misacylated-tRNA.
