Mechanisms of sigma-factor-dependent transcriptional control in Mycobacterium tuberculosis

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) causes 1.6 million tuberculosis (TB) related deaths per year, and current TB therapeutic strategies are becoming inadequate due to the emergence of drug resistant strains. One of the primary antibiotics used to treat TB infection acts at the level of transcription, inhibiting the RNA polymerase (RNAP). Sigma factors (σ) are accessory subunits that associate with the RNAP, directing a transcription- competent RNAP holoenzyme to unique genes. The σA RNAP holoenzyme is responsible for transcribing the majority of the Mtb genome, and as such, represents the major RNAP-dependent drug target. Of all the σs found in Mtb, only σA contains a N-terminal intrinsically disordered region (IDR) with strong charge segregation (i.e. clustering of positively/negatively charged residues). The propensity for disorder and charge segregation is conserved not only across mycobacteria, but more broadly across the whole Actinobacteria phylum, suggesting a functional, yet uncharacterized role of these σA IDRs in bacteria. This proposal seeks to understand the role of the Mtb σA IDR in transcriptional regulation, assessing what sequence and conformational features of the IDR encode functionality. Leveraging both experimental and computational approaches that overcome the technical challenges associated with the structural characterization of IDRs, I will assess how the conformational dynamics and interactions of the σA IDR impart transcriptional activity. I will employ a combination of in vitro kinetic measurements for transcriptional activity, single-molecule fluorescence techniques for measurements of IDR conformations and dynamics, and molecular dynamics simulations and cross-linking mass spectrometry for identifying IDR- mediated interactions within the RNAP holoenzyme. Measurements will specifically investigate the role of charge-segregated disordered regions and post-translational modifications and be performed under conditions that mimic intracellular ionic concentrations during Mtb infection and in the presence/absence of antibiotics. Given the broad conservation of sequence features, conclusions made will likely be applicable beyond Mtb, leading to a new paradigm for how bacteria use IDRs in transcription. The research and training objectives in this proposal will further the understanding of mechanisms of gene regulation unique to disease-causing bacteria, while enabling me to obtain additional technical training to foster the future generation of academic biomedical investigators.