Characterization of novel binding sites and regulatory activities for the transcription sigma factor, rpoN, in Salmonella
Bono, Ashley Christine
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During bacterial transcription, core RNA polymerase (ααββ’ω) transiently interacts with a σ factor to identify a promoter and then isomerizes into transcriptionally active open complex. Sigma54 (σ54 or RpoN) is the lone member of an alternative sigma factor family that is highly conserved across diverse bacterial species. Sigma54-RNA polymerase holoenzyme (Eσ54) is unique among bacterial holoenzymes, and similar to Pol II of eukaryotes, in its requirement for physical interaction with a protein activator and hydrolysis of ATP for isomerization into open complex. The focus of the original research presented in this dissertation is to define the global σ54 regulon in the model organism Salmonella enterica subspecies enterica serovar Typhimurium 14028s, and characterize the regulatory roles of σ54-dependent promoters and σ54 binding sites. Earlier work suggested that S. Typhimurium has a robust σ54-dependent regulon of diverse genes regulated by at least 13 bacterial enhancer binding proteins (bEBPs) that are each responsive to different environmental signals. To promote open complex formation by Eσ54 and stimulate expression of all σ54-dependent genes, a previously vetted, constitutively-active, promiscuous bEBP, DctD250 was expressed in wild-type and ΔrpoN strains. Transcriptome profiling and identification of σ54 DNA binding sites from immunoprecipitated σ54-chromosomal DNA (ChIP) were performed on tiling microarrays (chip). Three novel σ54-dependent transcripts, in addition to the previously predicted/known σ54-dependent transcripts, and 184 σ54 intergenic and intragenic DNA binding sites were defined. Thirteen Eσ54/σ54 binding sites, identified by ChIP-chip, were confirmed using electrophoretic mobility shift assays, revealing two novel sites that bind σ54 alone, as well as Eσ54, and provide insight into DNA sequence features that impact σ54 binding. Selected novel σ54-DNA binding sites and associated transcripts were further characterized by quantitative reverse-transcriptase polymerase chain reaction. From the perspective of recently published transcription factor regulon studies in S. Typhimurium and Escherichia coli, these results suggest regulatory mechanisms for σ54 binding site that integrate the σ54 regulon into the complex network of transcription factor regulons controlling the cellular response to the myriad of stressors that it encounters during the infection process.