Type of Document	Dissertation
Author	Perera, Inoka Chinthana
URN	etd-07082010-164601
Title	Urate Responsive MarR Homologs
Degree	Doctor of Philosophy (Ph.D.)
Department	Biological Sciences
Advisory Committee	Advisor Name Title Grove, Anne Committee Chair Ding, Huangen Committee Member Newcomer, Marcia Committee Member Waldrop, Grover L. Committee Member Williams, Cathleen Dean's Representative
Keywords	oxidative stress transcriptional regulator urate MarR
Date of Defense	2010-06-17
Availability	restricted
Abstract Differential gene expression in response to internal and external stimuli is studied in detail to understand the intricate mechanisms underlying response to various environmental stressors in microorganisms. MarR family transcriptional regulators have been studied for their involvement in such mechanisms. This work elucidates the mechanism of urate-induced attenuation of DNA binding of HucR, a MarR homolog, and extends this mechanism to describe a novel subfamily of MarR homologs responsive to urate, proposing a physiological relevance of utilizing urate as a signaling molecule. HucR (hypothetical urate regulator) binds to the shared promoter region between uricase and hucR genes. It has high specificity for urate in attenuation of DNA binding. The ligand-binding site in HucR was identified using molecular-dynamics guided mutational analysis, leading to a proposed mechanism for the attenuation of DNA binding upon interaction of urate. According to this model, urate is anchored in the binding pocket by W20 and R80 while a charge-repulsion displaces D73, which propagates the conformational change to the DNA recognition helix. A possible extension of this mechanism to other MarR homologs was examined through homology search where a number of MarR homologs were identified as conserving the residues involved in urate binding. Further, they show high sequence identity in helix-3, which includes the conserved aspartic acid residue and in the DNA recognition helix, a sequence conservation that correlates to the conservation of bases in their proposed 18 bp consensus dyadic-binding site. To further investigate this phenomenon, Agrobacterium tumefaciens-encoded PecS, which conserves these residues, was studied in detail. PecS binds to the shared promoter region between the genes pecS and pecM while urate attenuates DNA binding in vitro and elevates the transcript levels in vivo. This study thus identifies a novel subfamily of MarR family transcription factors that bind urate and proposes a novel signalling function of urate, wherein invading bacteria utilize urate produced by the host to promote successful host colonization.
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