Abstract
The regulation of ribosomal RNA (rRNA) is closely tied to nutrient availability, growth phase, and global gene expression, serving as a key factor in bacterial adaptability and pathogenicity. Mycobacterium tuberculosis (Mtb) stands out from other species with a single ribosomal operon controlled by two promoters: rrnAP3 and rrnAP1 and a high ratio of sigma (σ) factors to genome size. While the primary σ factor σA is known to drive ribosomal transcription, the alternative σ factor σB has been proposed to contribute to the transcription of housekeeping genes, including rRNA under a range of conditions. However, σB's precise role remains unclear. Here, we quantify steady-state rates in reconstituted transcription reactions and establish that σA-mediated transcription from rrnAP3 dominates rRNA production by almost two orders of magnitude with minimal contributions from σB holoenzymes and/or rrnAP1 under all conditions tested. We measure and compare the kinetics of individual initiation steps for both holoenzymes which, taken together with the steady-state rate measurements, lead us to a model where σB holoenzymes exhibit slower DNA unwinding and slower holoenzyme recycling. Our data further demonstrate that the transcription factors CarD and RbpA reverse or buffer the stimulatory effect of negative superhelicity on σA and σB holoenzymes respectively. Lastly, we show that a major determinant of σA's increased activity is due to its N-terminal 205 amino acids. Taken together, our data reveal the intricate interplay of promoter sequence, σ factor identity, DNA superhelicity, and transcription factors in shaping transcription initiation kinetics and, by extension, the steady-state rates of rRNA production in Mtb.