Ribosomal RNA methylation by GidB modulates discrimination of mischarged tRNA
- Center for Global Health and Infectious Disease, Tsinghua University School of Medicine, China
- School of Life Science, Tsinghua University, China
- Division of Experimental Medicine, University of California, San Francisco, United States
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, United States
- Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, United States
Figures
Figure 1 with 2 supplements
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 HWS19 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 HWS19 measured by Renilla–Firefly dual-luciferase reporter as depicted in C. (E) gidB mutation was mapped onto three candidates by whole genome sequencing (*p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test).
Figure 1—figure supplement 1
Susceptibility to streptomycin of WT and HWS19.
HWS19 is more susceptible to MIC of streptomycin than WT. Data are presented as means of three biological replicates ± SD. **p < 0.01 by t-test.
Figure 1—figure supplement 2
Mistranslation level of the other suppressor candidates with gidB mutations.
N to D mistranslation rates were measured in seven suppressor candidates compared with wild-type and HWS19 by Renilla–Firefly dual-luciferase reporter. Mutations in each suppressor candidate are shown in Supplementary Information Table A.
Figure 2 with 3 supplements
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).
Figure 2—figure supplement 1
gidB deletion and complementation in WT and HWS19.
(A) Homologous recombination was performed for deletion of gidB, replaced with zeocin resistance marker. gidB complement was integrated into Giles site with pml1357. (B) PCR screening to verify successful deletion of gidB in both wild-type background and HWS19 background. Primer 1 (P1) is upstream of gidB, primer 2 (P2) is inside of gidB, and primer 3 (P3) is inside of zeocin resistance marker. See Supplementary file 1c for sequences of primers. Red box means successful deletion of gidB. (C) RT-qPCR to further verify the gidB mRNA level in gidB deletion and complementation strains. The gel image in (B) shows the DNA loaded from the PCR reactions to verify successful knock-out and is not cropped. The raw image, usually required for publication in eLife, is not available since it was lost when the Javid lab at Tsinghua University was shut down during the 2020 Covid pandemic.
Figure 2—figure supplement 2
gidB deletion increases translation fidelity in high mistranslation mutant.
E to Q mistranslation measured by gain-of-function dual-fluorescent reporter (Figure 2B) in high mistranslation HWS19 strain (A) and wild-type strain (B).
Figure 2—figure supplement 3
Gating strategy of flow cytometry and histogram of population count relying on GFP/RFP ratio.
(A) Gating M. smegmatis based on the estimation of the size and granularity as P1 in terms of SSC-A (X axis) and FSC-A (Y axis). (B) Gating single cell as P2 in terms of FSC-A (X axis) and FSC-H (Y axis). (C) Gating single cell again as P3 in terms of SSC-A (X axis) and SSC-H (Y axis). (D) Gating smegmatis expressing positive RFP as P4 in terms of RFP (X axis). (E) Histogram of population count (Y axis) relying on GFP/RFP ratio (X axis). GidB deletion strains and complementation strains in high mistranslating background (F) and wild-type background (G). X axis is the value of GFP and RFP ratio, representing the Q to E mistranslation level. The strain distributes to the right presents a higher mistranslation level than the one to the left.
Figure 3 with 2 supplements
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’s t-test).
Figure 3—figure supplement 1
Deletion of GidB does not affect growth rate in axenic culture.
WT Msm and ΔgidB (A) and strain HWS19 and HWS19ΔgidB (B) and their respective complemented strains were grown in standard supplemented 7H9 medium, and optical density monitored over time. Points represent means ± SD.
Figure 3—figure supplement 2
Deletion of GidB does not affect translation rate of Nluc luciferase.
WT Msm and ΔgidB (A) and strain HWS19 and HWS19ΔgidB (B) were transformed with a tetracycline-inducible Nluc luciferase construct. Nluc activity was monitored over time as a proxy for translation rate in all four strains. Time = 0 represents time of addition of Atc (50 ng/ml). Experiment was performed using three independent biological replicates. Points represent means ± SD. Chloramphenicol (200 µg/ml) was used as a translation inhibitor control.
Figure 4 with 1 supplement
Deletion of GidB decreases mistranslation mediated by non-physiological ‘misacylated-tRNA’.
(A) The anticodon of an alanine-tRNA is mutated to tryptophan codon (EMAW) resulting in mistranslation from tryptophan to alanine (right) to mimic non-physiological misacylated-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’s t-test).
Figure 4—figure supplement 1
Corrected Renilla luciferase activity.
Renilla-WT_Firefly and Renilla-A214W_Firefly were transformed into wild-type smegmatis. The absolute ratio of Renilla-WT/ Firefly and Renilla-A214W/ Firefly was illustrated in the graph.
Figure 5
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’s t-test).
Figure 6 with 1 supplement
Structures of mycobacterial ribosomes with and without GidB-mediated methylation.
Ribosomal structures from the following Mycobacterium smegmatis strains: 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 = 146,688, resolution = 2.47 Å) and (D) zoomed-in site of the GidB methyltransferase target residue (G507).
Figure 6—figure supplement 1
Subunit profiling of different M.smegmatis strains.
Sucrose gradient profile of HWS19, HWS19-ΔgidB.
Figure 7
Structures of mycobacterial ribosomes with and without GidB-mediated methylation.
(A) HWS-19: Different views of the Msm-70S structure (particle number = 108,034, resolution = 3.26 Å) and (B) zoomed-in site of the GidB methyltransferase target residue (G507) and interacting residues. (C) HWS-19-ΔgidB: Different views of the Mycobacterium smegmatis 70S ribosomal structure (particle number = 136,418, map resolution = 3.07 Å) and (D) zoomed-in site of the GidB methyltransferase target residue (G507) and interacting residues.
Figure 8
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 loosened ribosome conformation compared to HWS19-ΔGidB ribosome, which translation is error-prone. HWS19-ΔGidB ribosome restores structural integrity could better discriminate against misacylated-tRNA.
Tables
Table 1
Cryo-EM data collection, refinement, and validation statistics.
| WT strain WT (EMDB-44092) (PDB 9B1Y) | WT strain gidB mutant (EMDB-44097) (PDB 9B24) | HWS19 strain WT (EMDB-44090) (PDB 9B1W) | HWS19 strain gidB mutant (EMDB-44091) (PDB 9B1X) | |
|---|---|---|---|---|
| Data collection and processing | ||||
| Magnification | 96,000 | 105,000 | 130,000 | 130,000 |
| Voltage (kV) | 300 | 300 | 300 | 300 |
| Electron exposure (e−/Å2) | 60.74 | 901.165 | 72.704 | 72.704 |
| Defocus range (μm) | 400–1400 | 500–1500 | 500–1500 | 500–1500 |
| Pixel size (Å) | 0.833 | 0.833 | 0.662 | 0.664 |
| Symmetry imposed | P1 | P1 | P1 | P1 |
| Initial particle images (no.) | 704,502 | 1,636,851 | 254,392 | 417,089 |
| Final particle images (no.) | 591,747 | 1,466,881 | 108,034 | 136,418 |
| Map resolution (Å) FSC threshold | 3.47 0.143 | 2.47 0.143 | 3.26 0.143 | 3.07 0.143 |
| Map resolution range (Å) | inf-3.47 | inf-2.47 | inf-3.26 | inf-3.07 |
| Refinement | ||||
| Initial model used (PDB code) | 5o60, 505j | 5o60, 505j | 5o60, 505j | 5o60, 505j |
| Model resolution (Å) FSC threshold | 3.32 0.143 | 3.05 0.143 | 4.22 0.143 | 3.69 0.143 |
| Model resolution range (Å) | inf-3.47 | inf-2.47 | inf-3.26 | inf-3.07 |
| Map sharpening B factor (Å2) | N/A | N/A | N/A | N/A |
| Model composition Non-hydrogen atoms Protein residues Ligands | 146,981 5723 629 | 146,980 5723 629 | 144,760 5877 629 | 144,818 5877 629 |
| B factors (Å2) Protein Ligand | 140.30 142.41 | 197.35 162.55 | 139.43 171.92 | 191.74 154.91 |
| R.m.s. deviations Bond lengths (Å) Bond angles (°) | 0.014 0.893 | 0.004 0.670 | 0.003 0.541 | 0.010 0.650 |
| Validation MolProbity score Clashscore Poor rotamers (%) | 2.72 59.82 0.86 | 2.08 19.66 0.13 | 1.80 9.34 0.40 | 1.70 7.56 0.10 |
| Ramachandran plot Favored (%) Allowed (%) Disallowed (%) | 92.44 7.47 0.09 | 95.78 4.17 0.05 | 95.56 4.36 0.09 | 95.82 4.11 0.07 |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/102752/elife-102752-mdarchecklist1-v1.docx
-
Supplementary file 1
Supplementary Information Tables A-C.
- https://cdn.elifesciences.org/articles/102752/elife-102752-supp1-v1.docx
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Ribosomal RNA methylation by GidB modulates discrimination of mischarged tRNA
eLife 13:RP102752.
https://doi.org/10.7554/eLife.102752.3
