Date of Award

Summer 8-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Mycobacterium tuberculosis is the causative agent of the disease tuberculosis (TB) and remains one of the deadliest microorganisms on the planet. The effort to eradicate M. tuberculosis would benefit from the development of novel therapeutics, which requires a detailed understanding of M. tuberculosis physiology. Like all living organisms, M. tuberculosis gene expression requires transcription. Transcription in the phylum Actinobacteria, which includes mycobacteria, is unique because it includes RNA Polymerase Binding Protein A (RbpA) that is essential in both M. tuberculosis and the nonpathogenic model organism Mycobacterium smegmatis. RbpA increases the housekeeping A and housekeeping like B interactions with the RNA polymerase (RNAP) and can increase transcription by both A and B bound RNAPs in vitro, suggesting that RbpA activates M. tuberculosis transcription. During transcription initiation, the equilibrium between the melted and unmelted promoter conformations is a common regulatory target. RbpA stabilizes the melted DNA conformation called the RNA polymerase open promoter complex (RPo). Structural studies revealed that RbpA is comprised of four structural domains including the N-terminal tail (NTT), core domain (CD), basic linker (BL) and sigma interaction domain (SID). RbpA BL interacts with the DNA phosphate backbone of the non-template strand while the SID mediates RbpA’s interaction with σA and σB. The activities of both the BL and SID are important for RbpA RPo stabilizing activity in vitro. Using a panel of RbpA point mutants and RbpA domain truncation mutants, I further characterized the activities of RbpA’s four structural domains in vitro and in vivo. The activities of all four domains are required for M. tuberculosis growth while only the BL and SID are required for M. smegmatis growth. RNA-sequencing analysis revealed that RbpA activates transcription of some genes while repressing the transcription of other genes, and the activities of the BL/SID and NTT/CD affect transcription of two distinct gene subsets. We determined that the SID is necessary and sufficient for RbpA interaction with both the A and B bound RNAPs and weakening RbpA’s interaction with the RNAP decreases RbpA protein levels in M. smegmatis. In vitro analysis done in collaboration with the Galburt lab revealed that the BL and SID are required for RbpA’s RPo stabilizing activity while the NTT and CD antagonize RbpA RPo stabilizing activity. Structural studies show that the NTT and CD are positioned near multiple RNAP-A holoenzyme functional domains, suggesting that the RbpA NTT and CD could have a number of effects on RNAP activity. However, these studies did not identify which contacts between the NTT or CD and the RNAP mediate the antagonism of RPo stability that we observed in our studies. In addition, structural studies predict that the RbpA NTT contributes contacts to the binding site for the antibiotic fidaxomicin (Fdx) on the RNAP. Deletion of the NTT results in a decrease in M. smegmatis sensitivity to Fdx, but whether this is caused by a loss of contacts with Fdx was unknown. Using a panel of rbpA mutants with single amino acid substitutions replacing conserved residues within the NTT, I probed what RbpA NTT residues are involved in regulating Fdx activity and RPo stability. We identify multiple residues in the NTT along with other RbpA domains that contribute to Fdx activity in vivo. We also identify RbpA NTT residues that contribute to antagonism of RbpA-mediated stabilization of RPo and link this antagonism to increased full length transcript production. In work characterizing the role of RbpA’s interaction with B I determined that the loss of RbpA BL or SID activities alters sigB from its typical status as a non-essential gene to a synthetically essential gene. RNA-sequencing analysis of M. smegmatis with a sigB deletion (sigB) shows that sigB regulates a cohort of transcripts that if translated encode short and highly charged proteins. In addition, the subset of transcripts differentially expressed in M. smegmatis sigB shares little overlap with the gene subset differentially expressed in M. smegmatis expressing rbpA with a point mutation in the SID that weakens RbpA interaction with both A and B, suggesting that RbpA-independent B regulation occurs during logarithmic growth. My thesis work has improved our understanding of RbpA regulation of mycobacteria transcription and RbpA’s role in fidaxomicin activity. This work shows that RbpA regulates transcription through novel mechanisms, shedding new light on the similarities and differences between the Actinobacteria and E. coli paradigms of bacterial transcription. Furthermore, the design of future therapeutics might benefit from this interrogation of RbpA activities and how this essential protein contributes to Fdx activity against mycobacteria.

Language

English (en)

Chair and Committee

Christina L. Stallings

Committee Members

Michael G. Caparon, Eric A. Galburt, Daniel E. Goldberg, Hani S. Zaher,

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