Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Mitchell Lewis

Abstract

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.

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