Role of the unstructured N-terminal region and interacting proteins on the function of Mycobacterium tuberculosis σA

Abstract

A distinctive feature of the bacterial RNA polymerase is the modular organization. The core of this enzyme, comprised of a dimer of the α subunit assembles with the β, β', and ω subunit. The core RNA polymerase enzyme derives promoter recognition features by virtue of its reversible association with multiple σ factors. This aspect, wherein the RNA polymerase is recruited to a target promoter by a competition between multiple σ factors, each recognizing a specific promoter, led to the so-called ‘partitioning of σ factor space’ model of bacterial transcription. The relative intracellular levels of different σ factors are significant in this model as these σ factors compete for RNA polymerase binding. The intracellular levels and localization of different σ factor levels are well characterized involving transcription, translation, and post-translation mechanisms. Surprisingly, little was known about the mechanism(s) that regulates the intracellular level or activity of the principal σ factor, σA The work reported in this thesis thus focused on addressing this lacuna in the context of the primary essential σ factor, Mycobacterium tuberculosis σA. One of the aspects investigated was the N-terminal region of σA in M. tuberculosis, examining its role in promoter binding and transcription. Depletion of σA results in a severe survival defect, and RNA-seq analysis indicates that σA regulates a significant portion of the transcriptome. The study reveals that specific disordered residues in the N-terminal region are essential for σA function and that phosphorylation plays a regulatory role. Another aspect describes the proteins interacting with M. tuberculosis σA and their functional implications. Surprisingly, proteins associated with translation, such as IF3, S6, and S9, are identified as interacting partners. These interactions affect transcript levels and suggest a role in regulating intracellular σA levels. The other feature of M. tuberculosis RNAP-σA presents preliminary work on designing non-competitive modulators against M. tuberculosis transcription machinery targeting a conserved σA-RNA polymerase β’ protein-protein interface is designed and synthesized. These modulators show the potential to both increase and decrease transcript levels, offering insights into the transcription mechanism in M. tuberculosis. Together, the work described in this thesis illustrates the multiple regulatory mechanisms that could govern the intracellular levels of free active σA and the role of the distinctive N-terminal intrinsically disordered region in this transcription factor. These studies thus lay the framework for more directed approaches to target this essential transcription factor in this slow-growing human pathogen.