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.