ORCID

http://orcid.org/0000-0001-6159-1607

Date of Award

Spring 5-15-2023

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Plant & Microbial Biosciences)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Mycobacterium tuberculosis (Mtb) is an important human pathogen that is responsible for over 1 million deaths each year. As a bacterial pathogen, Mtb is highly adapted to survival within the host environment, which imposes numerous environmental stresses on the bacteria. To survive these challenges, Mtb utilizes a network of transcription factors to adapt its gene expression and physiology. CarD is an essential transcription factor in mycobacteria that binds directly to RNA polymerase (RNAP) rather than DNA sequence motifs on the chromosome. Prior mechanistic studies of CarD’s function in mycobacteria have been limited to studies of CarD’s effects on individual kinetic rate constants in a handful of promoter contexts. However, chromatin-immunoprecipitation sequencing (ChIP-seq) studies indicate that CarD is present broadly across the mycobacterial genome. Therefore, to gain a holistic understanding of CarD’s role at all mycobacterial promoters, we performed RNA-sequencing (RNA-seq) on a set of Mtb and Mycobacterium smegmatis strains with point mutations causing defects in CarD’s function. Our study demonstrated that over 50% of the Mtb genome is transcriptionally responsive to CarD mutation and revealed a novel bi-functional role of CarD as both a transcriptional activator and a transcriptional repressor. I performed bioinformatic analyses to show that CarD-regulated promoters could not be defined by a specific DNA sequence motif but that the direction of regulation was correlated with promoter characteristics such as -10 element conservation and discriminator G+C%. Our RNA-seq dataset raised a new question – ‘How does CarD achieve regulatory specificity without binding specificity?’. In collaboration with the Galburt Lab, we developed a model in which the direction of CarD regulation is dictated by a promoter’s basal transcription initiation kinetics rather than transcription factor binding. Specifically, our model predicts that CarD would activate transcription from promoters that form an unstable promoter open complex (RPo) and repress transcription from promoters that form a stable RPo by inhibiting the step of promoter escape. To test this model, I performed in vitro transcription studies to determine CarD’s direct regulatory effect on a panel of promoters with varying levels of basal RPo stability. I showed that, indeed, while CarD activated transcription from the wild-type ribosomal RNA promoter Mtb rrnAP3, CarD repressed transcription from more stable promoter contexts. Furthermore, I showed that RPo stability and CarD regulation could be influenced by sequence-independent factors such as DNA supercoiling, revealing another dynamic of transcription regulation. Using the wealth of transcriptomic data that I gathered, I examined the overlap between CarD’s in vivo regulon with other known regulons to gain insight into CarD’s role in mycobacterial physiology. Although I could not identify any specific downstream transcriptional pathway that was uniformly differentially expressed, CarD’s regulon overlapped with numerous stress-responsive regulons including the DosRST two-component system and the alternative sigma factors σF, σH, and σM. My thesis work advances our understanding of how RNAP-binding bacterial transcription factors like CarD are capable of enacting specific transcriptional responses over a broad regulon. My research also highlights how a single transcription factor can exert multiple functions under different contexts including contexts that cannot be understood from DNA sequence alone. Lastly, my work on CarD may help inform future design of Mtb therapeutics by deepening our understanding of the transcriptional programs that Mtb uses to endure environmental stresses.

Language

English (en)

Chair and Committee

Christina L. Stallings

Committee Members

Petra A. Levin, Eric A. Galburt, Makedonka Mitreva, David Wang,

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