Dissecting the in vivo dynamics of transcription locking due to positive supercoiling buildup

Positive supercoiling buildup (PSB) is a pervasive phenomenon in the transcriptional programs of Escherichia coli. After finding a range of Gyrase concentrations where the inverse of the transcription rate of a chromosome-integrated gene changes linearly with the inverse of Gyrase concentration, we apply a Line Weaver-Burk plot to dissect the expected in vivo transcription rate in absence of PSB. We validate the estimation by time-lapse microscopy of single-RNA production kinetics of the same gene when single-copy plasmid-borne, shown to be impervious to Gyrase inhibition. Next, we estimate the fraction of time in locked states and number of transcription events prior to locking, which we validate by measurements under Gyrase inhibition. Replacing the gene of interest by one with slower transcription rate decreases the fraction of time in locked states due to PSB. Finally, we combine data from both constructs to infer a range of possible transcription initiation locking kinetics in a ch...正超螺旋积累（Positive supercoiling buildup, PSB）是大肠杆菌（Escherichia coli）转录调控过程中普遍存在的现象。我们在一系列促旋酶（Gyrase）浓度条件下发现，染色体整合基因的转录速率的倒数与促旋酶浓度的倒数呈线性相关，据此采用莱恩韦弗-伯克作图法（Line Weaver-Burk plot）解析了无PSB状态下的预期体内转录速率。随后，我们针对单拷贝质粒携带的同一基因，开展了基于延时显微镜技术（time-lapse microscopy）的单RNA生成动力学实验，验证了上述估算结果——该质粒载体已被证实不受促旋酶抑制的影响。接下来，我们估算了细胞处于锁定状态的时间占比以及锁定前的转录事件总数，并通过促旋酶抑制条件下的实验测量验证了该估算结果。将目标基因替换为转录速率更慢的基因后，因PSB引发的锁定状态时间占比会降低。最后，我们结合两种基因构建体的数据，推导出了一系列可能的转录起始锁定动力学，在一个……