RNA Polymerase Binding Protein A (RbpA) Regulation of Mycobacteria Transcription and Sensitivity to Fidaxomicin

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

Major alteration in coxsackievirus B3 genomic RNA structure distinguishes a virulent strain from an a virulent strain

Coxsackievirus B3 (CV-B3) is a cardiovirulent enterovirus that utilizes a 5′ untranslated region (5′UTR) to complete critical viral processes. Here, we directly compared the structure of a 5′UTR from a virulent strain with that of a naturally occurring avirulent strain. Using chemical probing analysis, we identified a structural difference between the two 5′UTRs in the highly substituted stem-loop II region (SLII). For the remainder of the 5′UTR, we observed conserved structure. Comparative sequence analysis of 170 closely related enteroviruses revealed that the SLII region lacks conservation. To investigate independent folding and function, two chimeric CV-B3 strains were created by exchanging nucleotides 104–184 and repeating the 5′UTR structural analysis. Neither the parent SLII nor the remaining domains of the background 5′UTR were structurally altered by the exchange, supporting an independent mechanism of folding and function. We show that the attenuated 5′UTR lacks structure in the SLII cardiovirulence determinant

Molecular dissection of RbpA-mediated regulation of fidaxomicin sensitivity in mycobacteria

RNA polymerase (RNAP) binding protein A (RbpA) is essential for mycobacterial viability and regulates transcription initiation by increasing the stability of the RNAP-promoter open complex (R

Domains within RbpA serve specific functional roles that regulate the expression of distinct mycobacterial gene subsets

The RNA polymerase (RNAP) binding protein A (RbpA) contributes to the formation of stable RNAP-promoter open complexes (R

Cooperative stabilization of Mycobacterium tuberculosis rrnAP3 promoter open complexes by RbpA and CarD

The essential mycobacterial transcriptional regulators RbpA and CarD act to modulate transcription by associating to the initiation complex and increasing the flux of transcript production. Each of these factors interacts directly with the promoter DNA template and with RNA polymerase (RNAP) holoenzyme. We recently reported on the energetics of CarD-mediated open complex stabilization on the Mycobacterium tuberculosis rrnAP3 ribosomal promoter using a stopped-flow fluorescence assay. Here, we apply this approach to RbpA and show that RbpA stabilizes RNAP-promoter open complexes (RP(o)) via a distinct mechanism from that of CarD. Furthermore, concentration-dependent stopped-flow experiments with both factors reveal positive linkage (cooperativity) between RbpA and CarD with regard to their ability to stabilize RP(o). The observation of positive linkage between RbpA and CarD demonstrates that the two factors can act on the same transcription initiation complex simultaneously. Lastly, with both factors present, the kinetics of open complex formation is significantly faster than in the presence of either factor alone and approaches that of E. coli RNAP on the same promoter. This work provides a quantitative framework for the molecular mechanisms of these two essential transcription factors and the critical roles they play in the biology and pathology of mycobacteria

Starvation sensing by mycobacterial RelA/SpoT homologue through constitutive surveillance of translation

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homolog (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomes in vivo remains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease-dependent manner. This loss is also observed in nonstarved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12 stalk base, suggesting that the aminoacylation status of A-site tRNA is surveilled during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with the ribosomes entering the translation cycle

Starvation Sensing by Mycobacterial RelA/SpoT Homologue Through Constitutive Surveillance of Translation

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homolog (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomes in vivo remains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease-dependent manner. This loss is also observed in nonstarved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12 stalk base, suggesting that the aminoacylation status of A-site tRNA is surveilled during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with the ribosomes entering the translation cycle

What is the difference between virulent and avirulent CVB3?

Coxsackievirus B3 (CVB3) is an enterovirus that is implicated in human diseases, most notably myocarditis. Similar to all other enteroviruses, CVB3 has a single stranded RNA genome that is typically broken into four regions. The four regions include the 5’untranslated region (5’UTR), the coding region, a 3’untranslated region (3’UTR) and a poly (A) tail. The 5’UTR function has been well studied and accepted as a critical element for CVB3 infection. The 5’UTR’s function in viral processes during infection is dependent on its structure. The 5’UTR must be folded into a specific set of structural domains. We have determined the secondary structure of the 5’UTR in both virulent (CVB3/28) and avirulent (CVB3/GA) strains of CVB3 and compared the structures. The virulent and avirulent 5’UTR secondary structures are dissimilar in a region that has previously been proven to be the CVB3 cardiovirulence determinant. This data strongly suggests the 5’UTR structure is a mechanism of CVB3 cardiovirulence

A Comparison of Genome Structures in Naturally Occurring Virulent and Avirulent Coxsackiesviruses

Coxsackievirus B3 (CVB3) in an enterovirus in the family Picornaviridae and relevant to human health, causing diseases including myocarditis, pancreatitis and aseptic meningitis. The viral genome includes a 742 nucleotide 5’Untranslated Region (5’UTR) that serves a critical role in CVB3 infection. Efficient viral replication and viral peptide synthesis require a specific 5’UTR secondary structure that has been extensively studied. Two naturally occurring strains of CVB3 include the virulent CVB3/28 and the avirulent CVB3/GA. Previous studies have used chemical probing to characterize the secondary structure of CVB3/28 and generate an experimentally supported structural model. This current study further examines and compares the CVB3 5’UTR secondary structure in both CVB3/GA and CVB3/28. The primary sequence of both strains have been determined in previous reports, and sequence changes occur at 63 of the 742 positions. Given this variability, the CVB3/GA 5’UTR may include mutations that disrupt or alter the formation of the specific secondary structures required for viral processes. Identification of 5’UTR structural differences between naturally occurring virulent CVB3/28 and avirulent CVB3/GA may provide a better understanding of the structures required for CVB3 5’UTR functionality as well as CVB3 virulence