Date of Award

1-1-2019

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (ix, 137 pages) : color illustrations.

Dissertation/Thesis Chair

Robert Osuna

Committee Members

Marlene Belfort, Richard Cunningham, Ing-Nang Wang

Keywords

CRP, dksA, ppGpp, RpoS, stringent response, transcription, Escherichia coli, Gene expression, Genetic transcription, Genetic regulation, RNA, Ribosomes

Subject Categories

Biochemistry | Microbiology | Molecular Biology

Abstract

DksA is a global transcription factor that binds RNAP directly to regulate the expression of many genes and operons, including ribosomal RNA, in a ppGpp-dependent or ppGpp–independent manner. It is also involved in facilitating the process of DNA replication by removing stalled transcription elongation complexes that could block the progress of the replication fork. In addition, DksA is important for colonization, establishment of biofilms, and pathogenesis. In order to sustain these various functions, an adequate level of cellular DksA is required. This work tested the hypothesis that the E. coli dksA is substantially regulated at the level of transcription. Using a combination of in vivo transcription assays, S1 nuclease mapping, and primer extension analysis, we identify three promoters (dksA P1, P2, and P3) that largely contribute to the transcription of dksA. In cells growing in rich medium, a previously identified dksA promoter (dksA P1) is highly transcribed during early and mid-exponential growth phase, and sharply shuts off thereafter. During late exponential growth phase, the relatively weaker, RpoS-dependent, dksA P2 and P3 promoters become stimulated. During this time dksA P2 is transiently stimulated while dksA P3 transcription continues well into the stationary phase. This results in high levels of dksA transcription during exponential growth phase and lower levels of transcription during stationary phase. We show that RpoS is required for the switch in promoter activity from dksA P1 to P2 and P3. In absence of RpoS, the promoter switch is not observed and transcription from the strong dksA P1 promoter persists into the stationary phase, despite the autoregulatory role of DksA on dksA P1 transcription. The nucleoid-associated protein Fis binds to five sites within the dksA P1 region represses transcription from this promoter. In absence of Fis, high levels of dksA P1 transcripts persist into the stationary phase, overriding the autoregulatory role of DksA on this promoter. These results indicate DksA autoregulation is not sufficient to fully control the expression of dksA P1. We confirm the negative regulation of DksA on dksA P1 and show that this effect is ppGpp-independent when cells are growing in nutritionally rich medium. Moreover, we show that DksA and ppGpp are both required for the stimulation of the dksA P3 and P2 promoters. Thus, like RpoS, DksA and ppGpp play a dual role in repressing dksA P1 and stimulating dksA P2 and P3 during the transition from exponential to the stationary growth phase.

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