CarD contributes to diverse gene expression outcomes throughout the genome ofMycobacterium tuberculosis

Abstract

The ability to regulate gene expression through transcription initiation underlies the adaptability and survival of all bacteria. Recent work has revealed that the transcription machinery in many bacteria diverges from the paradigm that has been established inEscherichia coli.Mycobacterium tuberculosis(Mtb) encodes the RNA polymerase (RNAP)-binding protein CarD, which is absent inE. colibut is required to form stable RNAP-promoter open complexes (RPo) and is essential for viability inMtb. The stabilization of RPoby CarD has been proposed to result in activation of gene expression; however, CarD has only been examined on limited promoters that do not represent the typical promoter structure inMtb. In this study, we investigate the outcome of CarD activity on gene expression fromMtbpromoters genome-wide by performing RNA sequencing on a panel of mutants that differentially affect CarD’s ability to stabilize RPo. In all CarD mutants, the majority ofMtbprotein encoding transcripts were differentially expressed, demonstrating that CarD had a global effect on gene expression. Contrary to the expected role of CarD as a transcriptional activator, mutation of CarD led to both up- and down-regulation of gene expression, suggesting that CarD can also act as a transcriptional repressor. Furthermore, we present evidence that stabilization of RPoby CarD could lead to transcriptional repression by inhibiting promoter escape, and the outcome of CarD activity is dependent on the intrinsic kinetic properties of a given promoter region. Collectively, our data support CarD’s genome-wide role of regulating diverse transcription outcomes.