Mechanism of Transcriptional Bursting in Bacteria

• A high-throughput single-molecule assay follows in vitro transcription in real time
• Positive supercoiling buildup inhibits transcription initiation and elongation
• Transcriptional bursting is dependent on the intracellular gyrase level
• Gyrase dissociation from and rebinding to a DNA loop cause transcriptional bursts

SummaryTranscription 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.

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