Regulation of Steady State Ribosomal Transcription in Mycobacterium tuberculosis: Intersection of Sigma Subunits,...

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 σ^A is known to drive ribosomal transcription, the alternative σ factor σ^B has been proposed to contribute to the transcription of housekeeping genes, including rRNA under a range of conditions. However, σ^B's precise role remains unclear. Here, we quantify steady-state rates in reconstituted transcription reactions and establish that σ^A-mediated transcription from rrnAP3 dominates rRNA production by almost two orders of magnitude with minimal contributions from σ^B 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 σ^B 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 σ^A and σ^B holoenzymes respectively. Lastly, we show that a major determinant of σ^A'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.