Student Theses and Dissertations
Date of Award
2024
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Elizabeth Campbell
Additional Thesis Advisor
Jeremy M. Rock
Keywords
transcription regulation, RNA polymerase, Mycobacterium tuberculosis, transcription factors, cell-free genomics, rifampicin resistance
Abstract
All life requires the expression of the genetic information stored in DNA into RNA, a process known as transcription. Hence, DNA-dependent RNA polymerases (RNAPs) perform the first step of gene expression, thereby dictating the amounts of gene products that proceed to downstream cellular reactions. The activity of RNAP can be modulated by numerous molecules including protein transcription factors (TFs), ligands, and antibiotics. Since bacteria only contain a single RNAP to express all genes, it is a prime therapeutic target. Most notably, the antibiotic rifampicin targets RNAP in the bacterial pathogen Mycobacterium tuberculosis (Mtb) and has therefore been a cornerstone of the frontline therapeutic regimen to treat tuberculosis (TB) since the 1960s. However, the rise of rifampicin-resistance necessitates new strategies to target RNAP and combat TB, which remains the leading cause of death from infectious disease worldwide. Efficient Mtb transcription requires multiple TFs that are essential for bacterial viability, pointing to specific steps of the transcription process that may be useful to target therapeutically. Yet, the direct genomic targets for most Mtb TFs are not known, limiting our understanding of Mtb gene expression. Obstacles include the lack of easily-predicted binding motifs, the degree of motif degeneracy tolerated, and the compensatory regulatory cascades triggered by perturbation of TFs in cells. In addition, the lack of tools to study non-model TFs, particularly those from difficult-to-culture microbes like Mtb, exacerbates these challenges. Specifically, we found that a major hindrance to identifying direct TF targets was the methodological gap between genomics and biochemistry. In cellulo genomics (e.g., ChIP-seq, RNA-seq) can provide genome-wide information, but pleiotropic indirect effects frequently obscure primary TF effects in cells, particularly when a TF is essential for viability or a global regulator. Conversely, in vitro transcription assays using purified RNAP can measure direct TF effects on RNA synthesis for a single gene, but these assays are too low-throughput for genome-scale transcription measurements and discovery of general principles. Therefore, the central aim of this thesis was the development of a novel “cell-free genomics” (CFG) method to bridge biochemistry and genomics. We reconstitute genome-wide transcription in vitro using purified components. We then quantify the output RNA using single-nucleotide-resolution RNA-seq and robust statistical analysis to count RNA 5' ends (to study transcription initiation) or RNA 3' ends (to study transcription elongation/termination) in the presence versus absence of essential TFs from tMb. CFG thus permits us to identify promoters and terminators whose expression is directly affected by TFs. We first validated CFG by counting the 5' ends of transcripts in the presence versus absence of the cyclic AMP receptor protein (CRP), the archetypal TF that originated the study of transcription regulation in 1970. CFG revealed 90 promoters where Mtb CRP alone is sufficient to modulate transcription initiation levels. From these direct promoter targets, we re-discover the known Mtb CRP binding motif and reveal that the predicted strength of CRP binding to its consensus site is a quantitative predictor of its effect size on a given promoter. We also identify known target genes found in cellulo. Integration of the CFG-derived “sufficiency regulon” of CRP with the "necessity regulon” previously determined using RNA-seq and ChIP-seq in exponentially growing Mtb cells revealed where CRP can act autonomously; where it requires other cellular regulators to modulate transcription; and where it exerts indirect transcriptional effects. Our interdisciplinary synthesis thus provides a roadmap to gain unprecedented resolution of transcription regulatory networks. We next applied CFG to identify promoters regulated by the actinobacteria-specific transcription initiation factor holo-WhiB1 in Mtb. Holo-WhiB1 was previously intractable by other approaches, and thus its direct genomic targets were unknown in any species. We first performed a whiB1 knockdown RNA-seq time course in Mtb cells, demonstrating its essentiality and revealing its global effects on the Mtb transcriptome. We next used CFG to identify the direct effects of holo-WhiB1 on transcription initiation genome-wide. Integration of in cellulo and cell-free hits permitted the identification of promoters that are directly regulated by holo-WhiB1 in exponentially growing Mtb cells, revealing that holo-WhiB1 activates numerous genes involved in translation and fatty acid biosynthesis. CFG revealed that unlike CRP, holo-WhiB1 does not appear to bind to a consensus motif at the position where it contacts promoter DNA (directly upstream of the housekeeping –35 promoter element). Rather, holo-WhiB1 activates transcription of promoters that have suboptimal –35 elements and represses transcription from promoters that have strong –35 contacts. We validate direct holo-WhiB1 activation and repression using in vitro transcription initiation assays with minimal promoter DNA constructs, allowing further mechanistic dissection of holo-WhiB1's effects using single-particle cryo-EM and functional mutagenesis. Lastly, we apply CFG to quantify the 3' ends of transcripts, permitting the first genome scale in vitro quantification of transcription termination in a cell-free system. We chose to study the essential pro-termination TFs Mtb NusA (conserved in all bacteria and archaea) and Mtb NusG (the only transcription factor conserved in all three domains of life), alone and in combination. Their mechanisms have remained elusive in part because neither has a predicted nucleic acid binding motif. We validate new NusA and NusG terminator targets in the Mtb genome using gold standard in vitro transcription termination assays and reveal a novel selectivity mechanism for how NusA and NusG regulate some terminators but not others. Specifically, we find distinct contacts between NusA versus NusG on pre-termination complexes likely favor distinct mechanisms of RNA release from the RNAP active site. NusG contacts both RNAP and DNA, but not RNA; therefore, NusG fails to stimulate terminators that have AT-rich downstream DNA and thus likely favor forward translocation of RNAP to stimulate RNA release. In contrast, NusA contacts both RNA and RNAP, but not DNA; therefore, NusA fails to stimulate terminators with RNA terminator hairpins that are predicted to invade the RNA–DNA hybrid and thus likely require a concomitant rotational wrenching of the hairpin away from RNAP to stimulate RNA release. Since these NusA/G contacts with transcription complexes are highly conserved, aspects of our model may generalize across cellular life. In sum, we propose that cell-free genomics comprehensively fills a critical methodological gap in the field of gene expression. CFG facilitates the study of TFs with various modes of action, including those intractable to study by other approaches, those lacking clear bioinformatic predictors like binding motifs, or both. CFG also holds promise for broader applications, including higher-order experimental designs, other transcriptional perturbations (e.g. ligands, antibiotics), and diverse species. By complementing existing approaches, CFG brings biochemistry and enzymology into the genomics era to transform our understanding of fundamental transcriptional regulation.
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Recommended Citation
Froom, Ruby, "Genome - Wide Regulatory Principles of Essential Transcription Factors from Mycobacterium Tuberculosis" (2024). Student Theses and Dissertations. 802.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/802
Comments
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy