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. ) stands out from other species with a single ribosomal operon controlled by two promoters: P3 and P1 and a high ratio of sigma (��) factors to genome size. While the primary �� factor �� is known to drive ribosomal transcription, the alternative �� factor �� has been proposed to contribute to the transcription of housekeeping genes, including rRNA under a range of conditions. However, ��'s precise role remains unclear. Here, we quantify steady-state rates in reconstituted transcription reactions and establish that ��-mediated transcription from P3 dominates rRNA production by almost two orders of magnitude with minimal contributions from �� holoenzymes and/or 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 �� 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 �� and �� holoenzymes respectively. Lastly, we show that a major determinant of ��'s increased activity is due to its N-terminal 205 amino acids. Taken together, our data reveal the intricate interplay of promoter sequence, �� factor identity, DNA superhelicity, and transcription factors in shaping transcription initiation kinetics and, by extension, the steady-state rates of rRNA production in .
