Allostery and Applications of the Lac Repressor

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Doctor of Philosophy (PhD)
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Biochemistry & Molecular Biophysics
autogenous switch
gene therapy
lac repressor
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The lac repressor has been extensively studied for nearly half a century; this long and complicated experimental history leaves many subtle connections unexplored. This thesis sought to forge those connections from isolated and purified components up to functioning lac genetic switches in cells and even organisms. We first connected the genetics and structure of the lac repressor to in vivo gene regulation in Escherichia coli. We found that point mutations of amino acids that structurally make specific contacts with DNA can alter repressor-operator DNA affinity and even the conformational equilibrium of the repressor. We then found that point mutations of amino acids that structurally make specific contacts with effector molecules can alter repressor-effector affinity and the conformational equilibrium. All results are well explained by a Monod, Wyman, and Changeux model of allostery. We next connected purified in vitro components with in vivo gene regulation in E. coli. We used an in vitro transcription assay to measure repressor-operator DNA binding affinity, repressor-effector binding affinity, and conformational equilibrium. Only the repressor-operator DNA binding affinity disagreed with literature values from other in vitro experiments, however it did agree with a published value which should hold under in vivo conditions. We were able to use our in vitro thermodynamic parameters to accurately predict the in vivo gene regulation when cell crowding was considered. Finally we developed an autogenously regulated lac repressor for AAV-mediated gene therapy. We were able to improve the gene regulation of the autogenous switch by using multiple operator DNA sites, a tetrameric lac repressor, and point mutations to the lac repressor. The autogenous switch was shown to function in various cell types and was capable of reversible regulation of luciferase in living mice.

Mitchell Lewis
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