Regulation of steady state ribosomal transcription in Mycobacterium tuberculosis: Intersection of sigma subunits, superhelicity, and transcription factors.

rRNA regulation in Mycobacterium tuberculosis (Mtb) is tightly linked to nutrient availability, growth phase, and global gene expression, influencing Mtb's adaptability and pathogenicity. Unlike most bacteria, Mtb has a single ribosomal operon with two promoters, rrnAP3 and rrnAP1, and a high ratio of sigma (σ) factors to genome size. While σ(A) is the primary driver of ribosomal transcription, σ(B) has been suggested to contribute under various conditions, though its role remains unclear. Here, we quantify steady-state transcription rates in reconstituted reactions and demonstrate that σ(A)-driven transcription from rrnAP3 dominates rRNA production, with minimal contributions from σ(B) or rrnAP1. Kinetic analysis suggests that σ(B) holoenzymes exhibit slower DNA unwinding and holoenzyme recycling. We also show that transcription factors CarD and RbpA reverse and buffer, respectively, the stimulatory effects of negative superhelicity on σ(A)-driven rRNA transcription. Finally, we identify the N-terminal 205 amino acids of σ(A) as a key determinant of its increased activity relative to σ(B). Our findings reveal the intricate interplay of promoter sequence, σ factor identity, DNA superhelicity, and transcription factors in shaping transcription initiation kinetics to ultimately influence rRNA production in Mtb.