Authors:

Andrew Diaz

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About Paper:

Operator Sites The E. coli lac repressor (LacI) regulates gene expression by binding to its base-sequence specific DNA operator while also interacting nonspecifically with genomic DNA. Although structural aspects of the LacI- DNA recognition pathway have been extensively studied, the detailed molecular mechanisms underlying base-sequence specific recognition remain incomplete. We aim to map the free energy landscapes that govern structural and dynamic fluctuations of defined DNA sequences, which enable LacI to recognize and tightly bind to its cognate operator site, and to slide and dissociate from non-operator sequences. We utilize fluorophore probes to site-specifically label DNA constructs at key positions to enable precise measurements of conformational changes upon protein binding, including critical regions such as the 'hinge region' that mediate regulatory dynamics. To study DNA breathing fluctuations, we employ a novel polarization-sweep single-molecule fluorescence method. Our experiments confirm that LacI forms a tightly-bound complex with optical probe labeled O1 DNA constructs and preliminary results indicate that fluorophore labels inserted at different operator positions experience distinct conformational effects upon LacI binding. These findings lay the groundwork for future studies to understand sequence-specific protein-DNA interactions and gene regulation, which can potentially provide new insights for the development of genome editing technologies. 133 UNIVERSITY OF OREGON • 2025 UNDERGRADUATE RESEARCH SYMPOSIUM TABLE OF CONTENTS

Source:

University of Oregon / 2025

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Co-authors:

Andrew Diaz