Soutenance de thèse : Raphaël FORQUET

Modelling transcriptional regulation by DNA supercoiling in bacteria

Doctorant : Raphaël FORQUET

Laboratoire INSA : MAP

Ecole doctorale : ED341  Évolution, Ecosystèmes, Microbiologie, Modélisation

Bacteria are exposed to environmental fluctuations, to which they respond by quick and global changes in gene expression. Usual models of transcriptional regulation are centred on transcription factors which recognise specific sequences in genes’ promoters, but disregard the important role of DNA supercoiling (SC), an ubiquitous property of the double-helix resulting from torsional stress. SC acts as a global and ubiquitous regulator in response to environmental changes, as suggested by many recent transcriptomics studies. The objective of the present thesis is to develop quantitative models of this regulation mode, by identifying the promoter-sequence determinants of gene SC-sensitivity, based on RNAP-DNA interaction and independently from additional regulatory proteins. To this end, we combine (i) the analysis of available transcriptomic data under conditions of SC variations, (ii) transcription assays on mutant promoters, and (iii) a thermodynamic modelling of transcription at the genome-scale. We first characterise the transcriptome of
D. dadantii, a phytopathogen in which extensive data have been accumulated regarding the role of SC during plant infection, defining its transcription units and promoters. We then present two models explaining how global SC variations can selectively activate/repress promoters, depending on (i) the G/C-content of their discriminator for SC- assisted promoter opening, and (ii) their spacer length for the SC-dependent orientation between -35 and -10 elements affecting RNA Polymerase binding. Transcription assays are conducted on mutant promoters, and quantitatively confirm the predictions of the models. The universality of these mechanisms is demonstrated by analysing transcriptomes of distant bacteria under conditions of SC variations. Altogether, these results show that SC, based on the fundamental properties of DNA, constitutes an ubiquitous regulation mode in the prokaryotic kingdom.