Role of the Discriminator Sequence in the Supercoiling Sensitivity of Bacterial Promoters

Abstract

In this study, we highlight the role of the discriminator as a global sensor of supercoiling variations and propose the first quantitative regulatory model of this principle, based on the specific step of promoter opening during transcription initiation. It defines the predictive rule by which SC quantitatively modulates the expression rate of bacterial promoters, depending on the G/C content of their discriminator and independently from promoter-specific regulatory proteins.

Quantitative contribution of the spacer length in the supercoiling-sensitivity of bacterial promoters

“…4B). This behaviour does not imply that the mechanism does not occur for this family of promoters, but possibly that its effects are over-come by a stronger opposite regulatory effect of SC at a later stage of transcription, in particular during open-complex formation where different elements of the promoter make extensive and complex contacts with RNAP, and the destabilisation of the double helix by SC has a drastic influence on the expression level [60, 16, 9].…”

“…S3. We previously showed [9] that the presence of the employed plasmids does not affect bacterial growth, and that results are not affected by plasmid copynumber variations (we compute relative expression levels between mutant promoters rather than absolute levels, and consistent observations were obtained in plasmid-borne or in chromosomal luciferase fusions), in agreement with other studies [39, 40]. The employed plasmids are well established as reflecting the average SC level of the chromosome [41], and also specifically in response to novobiocin-induced relaxation [42, 43, 44].…”

“…3, 17-nt spacers). These presumably reflect the modulation of promoter opening energy by the two opposite superhelical variations [9].…”

“…E. coli strain MG1655 cells were then transformed with these plasmids using a standard electroporation procedure. The following protocol is described elsewhere [9]. Briefly, E. coli cells carrying the plasmids with the different promoters were grown at 37°C in LB medium in a microplate reader (Tecan Spark).…”

“…At the mechanistic level, SC affects the transcription process at multiple steps, both indirectly through regulatory proteins, and directly by modulating the interaction of RNA Polymerase (RNAP) with DNA [3]. This includes the formation of the open-complex during transcription initiation, which is strongly facilitated by negative SC [7, 8] and plays a key role in the SC response of promoters [9]. But other steps of transcription also contribute to this response, including closed-complex formation [10], promoter escape [11], transcription elongation and termination [12].…”