Mechanism of Transcriptional Bursting in Bacteria
- Creators
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Chong, Shasha
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Chen, Chongyi
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Ge, Hao
- Xie, X. Sunney
Abstract
Transcription of highly expressed genes has been shown to occur in stochastic bursts. But the origin of such ubiquitous phenomenon has not been understood. Here, we present the mechanism in bacteria. We developed a high-throughput, in vitro, single-molecule assay to follow transcription on individual DNA templates in real time. We showed that positive supercoiling buildup on a DNA segment by transcription slows down transcription elongation and eventually stops transcription initiation. Transcription can be resumed upon gyrase binding to the DNA segment. Furthermore, using single-cell mRNA counting fluorescence in situ hybridization (FISH), we found that duty cycles of transcriptional bursting depend on the intracellular gyrase concentration. Together, these findings prove that transcriptional bursting of highly expressed genes in bacteria is primarily caused by reversible gyrase dissociation from and rebinding to a DNA segment, changing the supercoiling level of the segment.
Additional Information
© 2014 Elsevier. Under an Elsevier user license. Received 25 September 2013, Revised 17 March 2014, Accepted 8 May 2014, Available online 17 July 2014. We thank Xiaowei Zhuang for the collaboration on bacterial chromosomal structure study, which prompted us to conduct the current study; N. Patrick Higgins for providing the plasmid containing strong gyrase site sequence; Gene-Wei Li for development of FISH protocol; Minbiao Ji for help with microscope construction; Rahul Roy for advice on data analysis; and James Wang, Long Cai, Gene-Wei Li, and Paul Choi for critical reading of the manuscript. This work was supported by NIH Pioneer Award (1DP1OD000277; to X.S.X.), NIH grant TR01 (5R01GM096450-02; to X.S.X.), National Science Foundation of China (21373021; to H.G.), and the Foundation for the Author of National Excellent Doctoral Dissertation of China (201119; to H.G.). Author Contributions: X.S.X. conceived the project and supervised the experiments. S.C. developed the in vitro, single-molecule transcription assay. S.C. performed the in vitro imaging experiments, data analysis, and biophysical calculations based on the in vitro data. S.C. and C.C. performed the control of the enzyme activities in the in vitro, single-molecule transcription assay. C.C. performed the single-molecule mRNA FISH assay, data analysis, and live-cell experiments based on quantitative RT-PCR. C.C. made the DNA constructs for the in vitro, single-molecule assay and the FISH assay. H.G. built the mathematical model. H.G., C.C., and S.C fitted the model to the single-molecule FISH data. S.C., C.C., and X.S.X. designed the experiments and wrote the manuscript.Attached Files
Supplemental Material - 1-s2.0-S0092867414007399-mmc1.pdf
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Additional details
- PMCID
- PMC4105854
- Eprint ID
- 110711
- DOI
- 10.1016/j.cell.2014.05.038
- Resolver ID
- CaltechAUTHORS:20210902-233959965
- NIH
- 1DP1OD000277
- NIH
- 5R01GM096450-02
- National Science Foundation of China
- 21373021
- Foundation for the Author of National Excellent Doctoral Dissertation of China
- 201119
- Created
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2021-09-07Created from EPrint's datestamp field
- Updated
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2021-09-07Created from EPrint's last_modified field