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 chromosomal location, obtainable by tuning the transcription rate. We validate with measurements of transcription activity at different induction levels. This strategy for dissecting transcription initiation locking kinetics due to PSB can contribute to resolve the transcriptional programs of E. coli and in the engineering of synthetic genetic circuits.

正超螺旋积累（Positive supercoiling buildup, 简称PSB）是大肠杆菌（Escherichia coli）转录程序中普遍存在的现象。我们首先确定了一系列DNA旋转酶（Gyrase）浓度区间，在此区间内染色体整合基因的转录速率倒数与DNA旋转酶浓度的倒数呈线性相关；随后采用莱恩韦弗-伯克绘图法（Line Weaver-Burk plot）解析了不存在PSB时的预期体内转录速率。我们通过对单拷贝质粒携带的同一基因的单分子RNA生成动力学开展延时显微成像（time-lapse microscopy）验证了该估算结果——该质粒携带的基因已被证实不受DNA旋转酶抑制的影响。后续我们估算了细胞处于锁定状态的时间占比与锁定前发生的转录事件次数，并通过DNA旋转酶抑制实验验证了上述估算结果。将目标基因替换为转录速率更慢的基因后，因PSB导致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，推导得到了染色体位点上可通过调节转录速率获得的一系列可能的转录起始锁定动力学范围，并通过不同诱导水平下的转录活性测量结果完成了验证。这种解析PSB介导的转录起始锁定动力学的策略，有助于解析大肠杆菌的转录程序，并为合成遗传回路的工程化构建提供助力。