Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics

Abstract

ABSTRACTBacterial pathogens likeMycobacterium tuberculosis(Mtb) employ transcription factors to adapt their physiology to the diverse environments within their host. CarD is a conserved bacterial transcription factor that is essential for viability inMtb. Unlike classical transcription factors that recognize promoters by binding to specific DNA sequence motifs, CarD binds directly to the RNA polymerase (RNAP) to stabilize the open complex intermediate (RPo) during transcription initiation. We previously showed using RNA-sequencing that CarD is capable of both activating and repressing transcriptionin vivo. However, it is unknown how CarD achieves promoter specific regulatory outcomes inMtbdespite binding indiscriminate of DNA sequence. We propose a model where CarD’s regulatory outcome depends on the promoter’s basal RPostability and test this model usingin vitrotranscription from a panel of promoters with varying levels of RPostability. We show that CarD directly activates full-length transcript production from theMtbribosomal RNA promoterrrnAP3 (AP3) and that the degree of transcription activation by CarD is negatively correlated with RPostability. Using targeted mutations in the extended −10 and discriminator region of AP3, we show that CarD directly represses transcription from promoters that form relatively stable RPo. DNA supercoiling also influenced RPostability and affected the direction of CarD regulation, indicating that the outcome of CarD activity can be regulated by factors beyond promoter sequence. Our results provide experimental evidence for how RNAP-binding transcription factors like CarD can exert specific regulatory outcomes based on the kinetic properties of a promoter.