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| Status |
Public on May 15, 2024 |
| Title |
RNAP stalling-derived genome instability underlies ribosomal antibiotics efficacy and resistance evolution (ChIP-seq data) |
| Organism |
Escherichia coli |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Bacteria often evolve antibiotic resistance through mutagenesis. However, the processes causing the mutagenesis have not been fully resolved. Here we found that a broad range of ribosome-targeting antibiotics caused mutations through an underexplored pathway. Focusing on the clinically important aminoglycoside gentamicin, we found that the translation inhibitor caused genome-wide premature stalling of RNA polymerase (RNAP) in a loci-dependent manner. Further analysis showed that the stalling was caused by disruption of transcription-translation coupling. Anti-intuitively, the stalled RNAPs subsequently induced lesions to the DNA via transcription-coupled repair. While most of the bacteria were killed by genotoxicity, a small subpopulation acquired mutations via SOS-induced mutagenesis. Given that these processes were triggered shortly after antibiotic addition, resistance rapidly emerged in the population. Our work revealed a new mechanism of action of ribosomal antibiotics, illustrates the importance of dissecting the complex interplay between multiple molecular processes in understanding antibiotic efficacy, and suggests new strategies for countering the development of resistance.
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| Overall design |
To dissect the temporal relationship bewteen RNAP stalling and DNA damage, we profiled RNAP stalling and DNA damage using ChIP-seq on RpoB and Gam respectively. RpoB is the β subunit of RNAP; Gam is a phage protein that can capture the presence of DNA damage in vivo by binding to double-strand DNA ends. Briefly, we engineered a strain with IPTG-inducible 3xFLAG-tagged gam, supplemented 6 μg/ml of gentamicin to exponential phase bacteria (induced Gam expression ~3 h before gentamicin), and harvested the bacteria before and 20, 40, 60 min after gentamicin treatment respectively. We then split the samples in two halves, and performed ChIP-seq on RpoB and Gam-3xFLAG respectively
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| Contributor(s) |
Zheng Y, Chai R |
| Citation missing |
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| Submission date |
May 12, 2024 |
| Last update date |
May 15, 2024 |
| Contact name |
Ruochen Chai |
| E-mail(s) |
crc20@mails.tsinghua.edu.cn
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| Organization name |
Tsinghua University
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| Street address |
Haidian District
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| City |
Beijing |
| ZIP/Postal code |
100084 |
| Country |
China |
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| Platforms (1) |
| GPL25368 |
Illumina NovaSeq 6000 (Escherichia coli) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA1110738 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE267245_RAW.tar |
176.9 Mb |
(http)(custom) |
TAR (of TXT) |
| GSE267245_merged_gam_peaks.bed.gz |
2.7 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
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