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 (viii, 135 pages) : illustrations (chiefly color)

Dissertation/Thesis Chair

Robert Osuna

Committee Members

Marlene Belfort, Richard Cunningham, Cara Pager

Keywords

DksA, E. coli, fis, Phosphorylation, transcriptional regulation, uspA, Escherichia coli, Genetic regulation, Gene expression, Genetic transcription, RNA polymerases

Subject Categories

Biochemistry | Microbiology | Molecular Biology

Abstract

DksA is a bacterial gene regulator that functions synergistically with the stress alarmone ppGpp to mediate the stringent response. DksA also functions independently of ppGpp to regulate transcription of a number of genes. DksA function is dependent on its binding affinity to RNA polymerase and requires specific interactions between RNAP and catalytic amino acids located on the coiled coil tip, D74 and A76. While much of the previous work on DksA has focused on understanding the mechanisms of action and the numerous gene targets for transcriptional regulation, little is known about the mechanisms by which DksA expression and function may be regulated at the transcriptional and post-transcriptional levels. In this work we provide evidence from a variety of independent, experimental techniques that DksA is phosphorylated in vivo, and that an N-terminal phosphorylation, likely at amino acid K26, is required for the efficient stimulation of transcription from the uspA promoter. We were unable to identify a functional role associated with the additional phosphorylation site(s) disrupted by the ∆1-31 K101A, T102A, K104A, K105A mutant. In addition to phosphorylation, we probed for other effects of amino acid substitutions on a library of DksA mutants, including some affecting key residues known to be involved in ppGpp binding. We assayed for the ability to repress transcription of the fis promoter (fisp) or to stimulate transcription of uspA promoter (uspAp) in vivo. Our results show that DksA tolerates a large number of mutations with minimal effects on its ability to repress transcription in vivo, suggesting that the integrity and stability of the DksA structure may involve extensive intramolecular interactions not easily disturbed by point mutations. However, we also found several DksA mutations that strongly affect the ability to stimulate transcription but have little or no effect on its ability to repress transcription, demonstrating that certain functions of DksA are unique to its transcription stimulatory role. Some of these residues (R91, K94, K98, K139) are seen to be involved in ppGpp binding at the ppGpp site 2 of RNA polymerase, according to recent crystallographic evidence. Other residues not interacting with ppGpp are also required for transcription stimulation. Some interact with RNA polymerase in a different conformational state and others form 2 clusters in a solvent-exposed surface of DksA in the ternary complex, suggesting that interactions with other factors besides ppGpp are required for efficient transcription stimulation.

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