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 ribosomes suggest a role for GidB methylation in modifying contacts with Asn46.
A) The overall density map for the HWS19 WT ribosome is well resolved, permitting the assessment of conformation of the methylated state of G507. B) The overall density map of the HWS19 delta-gidB ribosome is also well resolved, indicating no major structural changes to the WT ribosome and permitting the assessment of the methylated state of G507. C) A zoom in of the site of GidB methylation (G507) reveals a potential contact between S12 Asn46 and G507. D) The unmethylated state created by delta-gidB lengthens the contact. The phenotypic recoding of S12 Asn46 to Asp in this background may disrupt this contact, leading to changes in translational fidelity.
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.
