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

Positive supercoiling buildup is a trigger of E. coli’s short-term response to cold shock

“…Soon after cells enter CS, the nucleoid area decreases for the entire period of our gene expression measurements (Figure 7A). Since past studies observed a similar phenomenon when inhibiting Gyrase [Palma et al, 2020] and since the nucleoid area is a good proxy for nucleoid density [Gray et al, 2019], whose increase is a common effect of PSB [Eriksson et al, 2002], we hypothesize that Gyrase duty cycles increase during CS. In agreement, the overlap between the Gyrase and nucleoid regions increase during CS (Figure 7D, see example cells), even though the ‘Gyrase region’ decreased in size relative to the nucleoid region (Figure 7B.1 and B.2).…”

“…If the origin of the reduction in transcription rates is supercoiling buildup, then it can be accounted for by an ON-OFF process (e.g., by replacing reaction 1.1 by reactions 1.3, Figure 6A) [Chong et al, 2014; Palma et al, 2020; Oliveira et al, 2016]. Else, if the reduction results from the slowdown of the forward kinetics of transcription initiation, e.g., due to an isomerization process preceding open complex formation (Buc and McClure, 1985), then it can be modeled by two forward, rate-limiting steps in RNA production (e.g., by replacing reaction 1.1 by reactions 1.2, Figure 6A).…”

“…Next, to investigate if genes with strong CSR also have high PSS, we also needed to classify genes as having ‘high’ |LFC NOVO |. For this, we considered that the inclination of the best fitting lines in Figure 6C likely differ with perturbation strengths (e.g., adding more than 50 μg/mL Novobiocin would cause stronger LFCs [Palma et al, 2020]). Since, on average, the response strength of CSR genes to 15 °C was twice as strong as their response to 50 μg/mL Novobiocin, we classified responses of |LFC NOVO | > 0.4 as ‘strong’ (if their p-value < 0.05).…”

Analytical kinetic model of native tandem promoters in E. coli

“…Closely spaced promoters may be more sensitive to supercoiling buildup than single promoters [53][54][55]. If so, it will be useful to extend the model to include these effects [26]. Using such model and measurements of expression by tandem promoters when subject to, e.g.…”

“…Meanwhile, models based on empirical parameter values suggest that collisions between two elongating RNAPs are rare (because events such as pausing or simultaneous initiations from both promoters are rare). Also, even if and when such collisions occur, they are unlikely to result in fall-offs since the RNAPs are moving at similar speeds and in the same direction [9][10] [26].…”