Date of Award
Doctor of Philosophy (PhD)
All cellular life depends on the precise regulation of its genome to adapt to environmental changes. This thesis focuses on exploring gene expression mechanisms at the level of transcription. Transcription is the process in which the enzyme RNA polymerase (RNAP) uses DNA as a template to generate nascent RNA. RNAP first binds to a regulatory sequence within the DNA called a promoter. The combined processes that occur at the promoter define transcription initiation, including promoter binding, promoter opening around the transcription start site, and initial synthesis of short RNA transcripts. Once RNAP escapes the promoter, it elongates the initial RNA transcript to generate a full-length RNA, terminating this process either with the assistance of transcription factors or upon encountering a regulatory sequence within the genome. Relative to transcription elongation and termination, initiation is rate-limiting, making the understanding of promoter-specific initiation kinetics key to defining transcriptional regulatory mechanisms.To understand the molecular mechanisms by which initiation kinetics are regulated, one must first be able to quantitate basal (i.e., un-regulated) transcription kinetics. I employed and developed a variety of in vitro, real-time, fluorescent-based methodologies to monitor both initiation intermediates as well as the overall steady-state rate of full-length RNA synthesis. Using stopped-flow kinetics with fluorescent-labeled promoter DNA templates, I quantitated the kinetics of promoter binding, promoter opening, initial transcript synthesis, and promoter escape. By monitoring the generation of RNA transcripts that contain an aptamer-fluorescent-dye pair, I also quantitated the steady-state rates of full-length transcript production. Performing both these pre-steady-state and steady-state experiments, I was able to measure basal initiation kinetics for the Mycobacterium tuberculosis (Mtb) σA and the E. coli σ70 containing RNAP holoenzymes, where calculations of the overall rate of transcript synthesis derived from my measurements of pre-steady-state initiation rate constants correlated with the steady-state rates measured experimentally. Regulation of transcription in Mtb can be carried out in distinct ways relative to E. coli. For instance, E. coli lacks the transcription factors CarD and RbpA, both of which are essential for Mtb physiology. In this work, I illustrated CarD exists in a weak monomer-dimer equilibrium, where the dimer species is likely not physiologically relevant based on known measurements of cellular CarD concentrations. Additionally, I showed that the effects on steady-state RNA synthesis rates in the presence of these transcription factors can be quantitively predicted by the factor’s effects on basal transcription initiation kinetics.These kinetic measurements obtained in the presence of CarD and RbpA also led us to propose a new regulatory model. CarD and RbpA can act at multiple steps of initiation, increasing the rate of promoter opening (by itself, a mechanism of transcriptional activation) and decreasing the rate of escape (by itself, a mechanism of transcriptional repression). To account for these observed kinetic changes, we suggested that CarD and RbpA could either activate or repress transcription depending on the rate limiting step in initiation, which changes depending on the sequence of the promoter. Using this approach, we found that our theoretical model provided an explanation for the in vivo regulation by these factors, as inferred from RNA-sequencing data. Additionally, I used this same conceptual framework to quantitatively illustrate how simple changes in promoter sequence context can lead to either activation or repression of transcription. Taken together, this work required many technical advances including development of new fluorescent-based methods and integration of kinetic models to predict and ultimately measure gene regulation. By using the Mycobacterium tuberculosis transcriptional machinery as a model system, I was able to define unique regulatory mechanisms specific to this pathogenic bacterium, mediated by transcription factors that lack DNA-binding specificity. The findings presented here lay the groundwork for a holistic understanding of gene regulation driven by a unified mechanism: basal initiation kinetics.
Chair and Committee
Michael Greenberg, Tomasz Heyduk, Andrea Soranno, Christina Stallings,
Jensen, Drake, "Fluorescent-based Approaches for Quantifying Bacterial Transcription Initiation Kinetics" (2023). Arts & Sciences Electronic Theses and Dissertations. 2865.
Available for download on Monday, April 22, 2024