CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis

Abstract

Significance Our understanding of the bacterial transcription machinery is largely based on studies in Escherichia coli. However, the human pathogen Mycobacterium tuberculosis (Mtb) and numerous other bacteria require an additional RNA polymerase binding protein called CarD, which is absent from E. coli, to form stable transcription initiation complexes. On the basis of CarD’s ability to stabilize transcription initiation complexes, it was predicted to function as a transcription activator. Here, we find that CarD serves as a global transcription regulator in Mtb that can either activate or repress gene expression, depending on the intrinsic properties of a given promoter. These studies elucidate paradigms of transcription regulation in the many bacteria phyla that encode homologs of CarD. 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 in Escherichia coli. Mycobacterium tuberculosis (Mtb) encodes the RNA polymerase (RNAP)-binding protein CarD, which is absent in E. coli but is required to form stable RNAP-promoter open complexes (RPo) and is essential for viability in Mtb. The stabilization of RPo by 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 in Mtb. In this study, we investigate the outcome of CarD activity on gene expression from Mtb promoters 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 of Mtb protein 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 RPo by 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.