Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Rahul M. Kohli

Abstract

Diversification of the genetic code in response to selective pressures can render organisms more fit to particular stresses. In many bacteria, the inducible prokaryotic DNA damage (SOS) response facilitates survival and adaptation to genotoxic stresses by upregulating genes involved in both high-fidelity and pro-mutagenic DNA damage repair. Within pathogenic bacteria, treatment with genotoxic antibiotics can induce the SOS response and lead to the acquisition of antibiotic resistance. Interest in disarming the SOS-dependent ability of bacteria to evade antibiotics has prompted investigation into the mechanisms underlying SOS pathway activation. Two proteins, the repressor LexA and the DNA recombinase RecA, operate together to regulate SOS gene expression. In response to DNA damage, RecA directly stimulates an autoproteolysis reaction within LexA that leads to SOS induction. Although this direct interaction is necessary, how complex formation results in autoproteolysis remains unknown. Here, we aimed to use the fluorescent unnatural amino acid acridonylalanine as a probe of the interaction between LexA and RecA. In this work, we first demonstrate how directing the evolution of a tRNA synthetase against the incorporation of unwanted contaminants can result in large increases in the selectivity of this enzyme for acridonylalanine. Recognizing that acridonylalanine incorporation may be poorly tolerated at certain positions in either LexA or RecA, we also describe a systematic evaluation of the effect of its incorporation at different positions on soluble protein expression. While acridonylalanine incorporation at different positions affects soluble protein expression, we could not determine any amino acid properties that reliably correlate with protein solubility. Finally, we show how a fluorescently-labeled LexA variant can be used to monitor the kinetics of association with RecA*. With this assay, we report the kinetic and thermodynamic parameters underlying the interaction of full-length LexA with RecA. Additionally, we provide direct evidence for a binding site on LexA for RecA. Altogether, the work presented here demonstrates how deliberately expanding the genetic code through scientific means enabled the design of new tools for studying protein function, and, in this case, allowed us to probe a protein-protein interaction that regulates a pathway naturally involved in diversifying the genetic code.

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