Date of Award
2026
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Elizabeth Campbell
Abstract
Tuberculosis (TB) is the leading cause of death worldwide due to an infectious agent. Although this is true globally, the greatest impact of TB is concentrated in regions of the world that are plagued by poverty and scarce resources. Mycobacterium tuberculosis (Mtb) is the bacterium that causes TB infection. Mtb is spread from person-to-person by coughing infectious droplets. These droplets carrying Mtb take residence in the lungs of an infected person, where it can persist even in the absence of active symptoms. Part of what makes TB such a successful pathogen is its ability to evade antibiotics. Currently, drug-sensitive TB is treated by a cocktail of 4 antibiotics over the course of 4- 6 months. However, drug-resistant TB calls for additional second and third-line antituberculars and is prescribed for a total of 6-9 months. These treatments are far from ideal and result in a wide range of unpleasant symptoms, such as gastrointestinal, neurological, and hepatic side effects. For these reasons, it is incredibly difficult for patients to successfully complete TB therapy, which further exacerbates antibiotic resistance. Approximately 400,000 people develop drug-resistant TB each year. Therefore, new drug targets are desperately needed. Considering this, we are studying the transcription cycle of Mtb, which is regulated differently than the well-studied model bacterium Escherichia coli (Eco). While transcription initiation is well characterized in the field, later steps like elongation, pausing, and termination remain underexplored. Here, we use structural, biochemical, and functional approaches to characterize later steps of the Mycobacterial transcription cycle. We aim to shed light on vulnerabilities within the cycle that may be targeted by therapeutics. We are therefore studying RNA polymerase (RNAP), the central enzyme of transcription, and associated transcription factors. We aimed to study pausing, an essential regulatory step occurring during elongation. Elongation is not constant but is instead intermittently interrupted by “pausing” events where the RNAP stops and waits for further instructions from the cell before resuming transcription. Pausing is regulated by transcription factors. One such factor is known as NusG- the only transcription factor conserved across all three domains of life. NusG was identified as an anti-pausing factor in Eco, meaning that it decreases pausing. Our studies have identified the previously unknown function of Mtb NusG. We conducted in vitro transcription assays, structural studies, and in vivo functional studies to reveal that Mtb NusG is a pro-pausing factor, opposite to Eco. This pro-pausing activity is modulated by a conformational change in the RNAP known as “swiveling”, involving the transition of the enzyme into an inactive state that is incompatible with nucleotide catalysis. Using single particle analysis, we show structurally how Mtb NusG stabilizes the swiveled state, promoting pausing, while Eco NusG stabilizes the “anti-swiveled” state, promoting elongation. Following this study, we sought to investigate the clinical relevance of pausing. Rifampicin is the most potent antitubercular used in the clinic. A single point mutation in the β subunit of RNAP, βS450L, accounts for ~70% of all Rifampicin-resistant (RifR) Mtb in the clinic. The presence of the βS450L mutation reduces transcription speed by 4-fold leading to over-pausing and over-termination relative to wild type (WT). This hyper-termination effect results in a fitness defect. However, there are secondary, compensatory mutations existing in the RNAP and other transcription factors that relieve hyper-termination effect and restore fitness to the cell. Using biochemical and functional, in vivo studies, we showed the first mechanism of RifR compensation in NusG. Utilizing a genome-wide association study, we discovered novel compensatory mutations in NusG. These mutations alleviate the fitness cost of βS450L and restore transcription speed close WT levels. Beyond NusG mutants, there are also compensatory mutations in RNAP subunits α and β’, or rpoA and rpoC, respectively that correct for RifR fitness defects. These are in fact the most common type of compensatory mutations. Following up on our work into RifR compensatory evolution in nusG, we are investigating the mechanisms by which the RNAP mutants rescue RifR fitness defects using structural and biochemical approaches. The data presented in this thesis both characterizes the structure and function of the universal transcription factor NusG as a pro-pausing factor in Mtb and also shows how the later transcription steps of pausing and termination could serve as targets for antituberculars. We show the first mechanism of RifR fitness compensation and the importance of maintaining ratios of elongation and termination for optimized bacterial fitness. Targeting pausing and termination and associated factors to enhance swiveling could exacerbate the fitness defect associated with RifR βS450L Mtb, combatting drug resistance. Similar compensatory mechanisms are known to exist in other subunits of RNAP, rpoA and rpoC. Understanding how these mutations work mechanistically could shed light on novel targets for inhibiting drug-resistant TB.
Recommended Citation
Delbeau, Madeleine, "Studies into Transcriptional Pausing Regulators and Drug Resistance in Mycobacterium Tuberculosis" (2026). Student Theses and Dissertations. 835.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/835
Comments
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy