Regulation of Transcription and Stress Response by CarD in Mycobacteria

Mycobacterium tuberculosis, the causative agent of Tuberculosis, infects over one third of the world\u27s population. To control this epidemic, we must develop new chemotherapeutic strategies for treatment, which requires further insight into the physiology of this bacterium. Previous studies have identified CarD as a transcriptional regulator essential during both acute and persistent infection. Depletion of CarD sensitizes strains to a diverse panel of stresses and deregulates several hundred genes and ribosomal RNA (rRNA) which suggested that CarD may be a transcriptional regulator. Chromatin immunoprecipitation sequencing experiments showed that CarD was localized to promoters throughout the genome, suggesting that CarD regulates transcription initiation. In collaboration with the Darst Lab, we published the first crystal structure of a CarD homolog. CarD\u27s N-terminal domain was homologous to a known RNA polymerase (RNAP) interacting domain and C-terminal domain with a novel fold. Modeling CarD onto initiation structures of RNAP position CarD\u27s C-terminal domain to interact with DNA. We identified three independent activities of CarD: binding RNAP, binding DNA, and the activity of a highly conserved tryptophan residue that we predict stabilizes the transcription bubble. Using a panel of single mutations in carD that attenuate one of these three activities, I characterized the roles of each of CarD\u27s activities in vivo and in vitro. All three of CarD\u27s activities are necessary for optimal growth, antibiotic resistance, stabilizing RNAP-promoter complexes, and activating transcription from rRNA promoters. This work contributed to a model in which CarD slows the rate of transcription initiation DNA bubble collapse and accelerates DNA opening. In further studies of CarD, I discovered a correlation between the cellular concentration of CarD and growth rate and showed that this growth rate dependence is not due to an effect on the rRNA content of the cell. This separated CarD\u27s effect on growth rate from its effect on rRNA content for the first time, which indicates that this growth defect is a result of deregulation of non-rRNA promoters. Additionally, I elucidated a new mechanism of regulating CarD activity through turnover of free protein. Most recently, I discovered the extent of CarD regulation in mycobacteria through RNA sequencing experiments. These studies revealed that more than 80% of the transcripts in the genome are significantly affected by alterations in CarD activity. Furthermore, there are transcript-dependent effects of CarD that imply that CarD is responsive qualities of the promoters but showed that the promoter sequence only partial explains this specificity. My thesis work has dramatically advanced our understanding of the mechanism of transcriptional regulation by CarD. CarD\u27s activity at transcription initiation complexes is an entirely novel mechanism of transcriptional regulation, creating a new paradigm of transcriptional regulation in prokaryotes. Furthermore, as CarD is conserved in many bacteria, its function has broad implications for bacterial transcription beyond mycobacteria. Finally, since CarD is essential in mycobacteria and absent from eukaryotes, my work will inform the development of new strategies to inhibit CarD activity as novel therapies to treat tuberculosis