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

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• Ribosomal RNA (rRNA) regulation in Mycobacterium tuberculosis (Mtb) impacts its adaptability and pathogenicity.
• Mtb possesses a unique single ribosomal operon with two promoters (rrnAP3 and rrnAP1) and multiple sigma (σ) factors.
• The precise roles of σ^A and σ^B in Mtb rRNA transcription remain incompletely understood.

Purpose of the Study:

• To quantify steady-state transcription rates of rRNA in Mtb.
• To elucidate the contributions of different promoters and sigma factors to rRNA production.
• To investigate the influence of DNA superhelicity and transcription factors on rRNA synthesis.

Main Methods:

• Reconstituted in vitro transcription assays.
• Kinetic analysis of transcription initiation and holoenzyme recycling.
• Characterization of transcription factor (CarD, RbpA) and DNA superhelicity effects.
• Identification of key domains within sigma factors.

Main Results:

• σ^A-driven transcription from the rrnAP3 promoter is the dominant pathway for Mtb rRNA production.
• σ^B holoenzymes exhibit reduced DNA unwinding and holoenzyme recycling efficiency compared to σ^A.
• CarD and RbpA modulate the impact of negative superhelicity on σ^A-dependent transcription.
• The N-terminal 205 amino acids of σ^A are crucial for its higher activity relative to σ^B.

Conclusions:

• Mtb rRNA production is predominantly controlled by σ^A at the rrnAP3 promoter.
• Distinct kinetic properties of σ^A and σ^B holoenzymes contribute to differential rRNA synthesis.
• Transcription factors and DNA topology play critical roles in fine-tuning rRNA production, influencing Mtb's response to environmental cues.