Regulation of Steady State Ribosomal Transcription in Mycobacterium tuberculosis : Intersection of Sigma Subunits, Superhelicity, and Transcription Factors

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: rrnA P3 and rrnA P1 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 rrnA P3 dominates rRNA production by almost two orders of magnitude with minimal contributions from σ ^B holoenzymes and/or rrnA P1 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 .