Abstract
Protein-DNA interactions are essential for gene replication and expression. Characterizing how proteins interact with and change the structure of DNA upon binding is crucial in elucidating the mechanism of protein function. Here, we use engineered DNA Holliday junction (HJ) as a single-molecule FRET reporter to study how CueR, a Cu(I)-responsive MerR-family metalloregulator, interacts with its DNA substrate for transcriptional regulation. By analyzing the single-molecule structural dynamics of the engineered HJ in the presence of varying concentrations of both apo- and holo-Cuer, we show how this metalloragulator interacts with and change the structures of the two HJ conformers, forming various protein-DNA complexes at different protein concentrations. We also show how apo- and holo-CueR differ in their interactions with DNA, as well as their similarities and differences with other members of the MerR-family of regulators, in particular in their mechanisms of switching off gene transcription after activation. This method of using engineered HJs to quantify changes in the structure and dynamics of DNA upon protein binding provides a new tool to elucidate the correlation of structure, dynamics, and function of DNA-binding proteins.
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