Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Cell. 2014 Jul 17;158(2):314-326. doi: 10.1016/j.cell.2014.05.038.
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.
高度表达基因的转录已被证明是以随机爆发的形式发生的。但是，这种普遍现象的起源尚不清楚。在这里，我们介绍了细菌中的这种机制。我们开发了一种高通量的体外单分子测定法，可实时跟踪单个 DNA 模板上的转录。我们表明，转录过程中 DNA 片段上正超螺旋的积累会减慢转录延伸的速度，最终停止转录起始。当拓扑异构酶与 DNA 片段结合时，转录可以恢复。此外，我们使用单细胞 mRNA 计数荧光原位杂交 (FISH)，发现转录爆发的占空比取决于细胞内拓扑异构酶的浓度。总之，这些发现证明了细菌中高度表达基因的转录爆发主要是由于拓扑异构酶与 DNA 片段的可逆解离和重新结合，从而改变了片段的超螺旋水平所致。
