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Allosteric regulation in AraC family transcriptional activators RhaS and RhaR
Klages, Joan E
Klages, Joan E
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Abstract
The AraC family of transcriptional activators is defined by the 99-amino acid long DNA binding domain (DBD) that contains two helix-turn-helix motifs. My work focuses on two of the family members RhaR and RhaS which together with cAMP receptor protein (CRP) are responsible for the transcriptional regulation of the L-rhamnose catabolic regulon in Escherichia coli. RhaR activates the rhaSR operon leading to increased expression of both activators, and RhaS activates the expression of the rhaBAD and rhaT operons which encode for the L-rhamnose catabolic enzymes and L-rhamnose import protein respectively.RhaR and RhaS in addition to the DBD also contain an N-terminal domain (NTD) that functions in binding the effector L-rhamnose and homodimer formation. The goal of this work is to further define the changes occurring in these two proteins upon L-rhamnose binding and how those changes impact the transcription activation of the regulated operons. This mechanism is complicated by the L-rhamnose binding within the NTD, not the DBD which contacts the DNA and the RNA polymerase molecule. Two hypotheses highlighting different potential changes occurring in these proteins have been proposed based on studies on RhaR and RhaS along with related proteins ToxT and AraC. The first proposes that changes within the DBD structure alter the ability of each monomer to bind to the DNA. The second asserts that a structural change impacting the overall homodimer structure could alter the ability of both monomers to bind simultaneously. The first type of change is proposed to be localized to the first HTH motif and the second type of change is proposed to occur at the dimerization interface.By measuring RhaR and RhaS activity at a single monomer binding site, as opposed to the full dimer binding site, I was able to isolate allosteric changes impacting the ability of each monomer to associate with the DNA. I found that the addition of L-rhamnose did not increase RhaR activation from half-sites but did increase RhaS activation from half-sites. These results suggest that rhamnose binding to RhaR impacts the structure of the homodimer, but not the monomer. In contrast, rhamnose binding to RhaS impacts the structure of each monomer - altering monomer affinity for the DNA sequence. Furthermore, using half-site sequences with substitutions I demonstrated that the first HTH in RhaS is likely key to the alterations in DNA affinity.Recently solved crystal structures of the RhaR NTD both with and without the bound L-rhamnose have highlighted two key areas undergoing structural changes between the two conditions. These include the opening of the effector binding pocket where L-rhamnose enters the protein and the dimerization interface. I investigated the role of the binding pocket opening in RhaR allosteric changes through an analysis of RhaR variants and identified five key residues within the region. Further mutational studies were completed to identify potential residue-residue interactions involving the identified residues at the binding pocket opening, but they were unsuccessful. Finally, I compared the changes in protein dynamics and homodimer forming interactions highlighting key differences between the two structures and implicating the dimerization interface as a possible location for allosteric changes in the RhaR dimer.In the last section of this work, I present the results of several RhaS mutant analysis studies identifying 26 residues that are likely involved in the allosteric changes occurring upon L-rhamnose binding within the RhaS NTD. The direct alignment of many of these identified residues to those also implicated in RhaR regulation, which based on current data is only regulated via dimer structural changes, highlights the possibility of dimer conformation changes also occurring in RhaS. Also, several of the residues identified in this screen align closely to those shown to be involved in AraC regulation but not in RhaR. These residues are possibly involved in HTH orientation changes which based on other findings in this work are likely occurring.
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Date
2023-01-01
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University of Kansas
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This item contains archived web content.
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Keywords
Microbiology, Genetics, Biochemistry, Allostery, AraC Family, L-rhamnose, RhaR, RhaS, Transcription activation