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 LineWeaver-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.

中文翻译：

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剖析了由于超螺旋堆积而导致的体内转录锁定动力学。

在大肠杆菌的转录程序中，正超螺旋堆积（PSB）是一种普遍现象。在找到一定范围的回旋酶浓度后，其中染色体整合基因的转录速率的倒数与回旋酶浓度的倒数线性变化，我们应用LineWeaver-Burk图来剖析在没有PSB的情况下预期的体内转录率。我们通过延时显微镜验证了单拷贝质粒携带的相同基因的单RNA产生动力学的估计，证实了该酶不受陀螺酶抑制作用的影响。接下来，我们估计锁定状态下的时间比例和锁定之前的转录事件数，这可以通过在回旋酶抑制下的测量来验证。用较慢的转录速率替换一个感兴趣的基因可减少由于PSB而导致处于锁定状态的时间比例。最后，我们结合来自两个构建体的数据来推断在染色体位置的一系列可能的转录起始锁定动力学，可以通过调节转录速率来获得。我们通过测量不同诱导水平下的转录活性来验证。剖析由于PSB而引起的转录起始锁定动力学的这种策略可以有助于解决大肠杆菌的转录程序，并有助于合成遗传电路的工程设计。我们通过测量不同诱导水平下的转录活性来验证。剖析由于PSB而引起的转录起始锁定动力学的这种策略可以有助于解决大肠杆菌的转录程序，并有助于合成遗传电路的工程设计。我们通过测量不同诱导水平下的转录活性来验证。剖析由于PSB而引起的转录起始锁定动力学的这种策略可以有助于解决大肠杆菌的转录程序，并有助于合成遗传电路的工程设计。