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    Molecular Mechanisms and Inhibition of Transcription Activation by Bacterial AraC Family Activator Proteins

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    KOPPOLU_ku_0099D_13093_DATA_1.pdf (3.939Mb)
    Issue Date
    2013-12-31
    Author
    Koppolu, Veerendra
    Publisher
    University of Kansas
    Format
    202 pages
    Type
    Dissertation
    Degree Level
    Ph.D.
    Discipline
    Molecular Biosciences
    Rights
    This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
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    Abstract
    AraC family proteins are transcriptional regulators that are defined by the presence of a conserved DNA binding domain (DBD). My research focused on three AraC family activators: Rns (activator of virulence genes in diarrhea-causing ETEC), VirF (activator of virulence genes in diarrhea-causing Shigella) and RhaR (activator of L-rhamnose catabolic operons in Escherichia coli). With the ultimate goal of discovery of novel antibacterial agents that inhibit the AraC family proteins, here I have investigated the molecular mechanism of transcription activation by Rns and RhaR. Site-directed mutagenesis of residues in the ETEC Rns N-terminal domain (NTD) identified three residues (N15, N16 and I17) that are required for the transcription activation function of Rns. Site-directed mutagenesis of residues in the Rns DBD (predicted to be contacted by the NTD residues) identified three residues (K216, Y251 and G252) that are required for transcription activation, and one residue (H250) that is required for both DNA binding and transcription activation. We propose that transcription activation by Rns involves contacts between RS2 and AS2 region residues and these contacts may impart the structure or dynamics required by Rns to activate transcription. In RhaR, I investigated the role of the RhaR Arm in transmission of the signal that effector (L-rhamnose) is bound from the NTD to the DBD, converting RhaR to its activating state. Site-directed mutagenesis results suggested that the RhaR Arm is involved in maintaining RhaR in its non-activating state. Our results suggest that residue L35 in the Arm makes inter-domain interactions with the RhaR DBD to reduce transcription activation by RhaR in the absence of L-rhamnose. To identify novel agents that target AraC family proteins, I tested the small molecule SE-1, which our lab identified as an effective inhibitor of the AraC family proteins RhaS and RhaR. Despite limited sequence identity, SE-1 was also shown to inhibit VirF and Rns activity in cell-based assays in E. coli. I showed that SE-1 blocked in vitro DNA binding by VirF and Rns, and expression of VirF-dependent virulence genes in Shigella. A collaborator showed that SE-1 inhibited invasion of Shigella into eukaryotic host cells. SE-1 did not detectably inhibit the growth or metabolism of the bacterial or eukaryotic host cells, respectively, indicating that the inhibition of invasion was not due to general toxicity. Overall, SE-1 appears to exhibit selectivity toward AraC family proteins, and has potential to be developed into a novel antibacterial agent.
    URI
    http://hdl.handle.net/1808/12999
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    • Dissertations [4321]
    • Molecular Biosciences Dissertations and Theses [273]

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    KU Libraries
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    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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