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: rrnAP3 and rrnAP1 and a high ratio of sigma ({sigma}) factors to genome size. While the primary {sigma} factor {sigma}A is known to drive ribosomal transcription, the alternative {sigma} factor {sigma}B has been proposed to contribute to the transcription of housekeeping genes, including rRNA under a range of conditions. However, {sigma}Bs precise role remains unclear. Here, we quantify steady-state rates in reconstituted transcription reactions and establish that {sigma}A-mediated transcription from rrnAP3 dominates rRNA production by almost two orders of magnitude with minimal contributions from {sigma}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 {sigma}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 {sigma}A and {sigma}B holoenzymes respectively. Lastly, we show that a major determinant of {sigma}As increased activity is due to its N-terminal 205 amino acids. Taken together, our data reveal the intricate interplay of promoter sequence, {sigma} factor identity, DNA superhelicity, and transcription factors in shaping transcription initiation kinetics and, by extension, the steady-state rates of rRNA production in Mtb.