title: Single-molecule study of metalloregulator CueR-DNA interactions using engineered Holliday junctions.
abstract: To maintain normal metal metabolism, bacteria use metal-sensing metalloregulators to control transcription of metal resistance genes. Depending on their metal-binding states, the MerR-family metalloregulators change their interactions with DNA to suppress or activate transcription. To understand their functions fundamentally, we study how CueR, a Cu(1+)-responsive MerR-family metalloregulator, interacts with DNA, using an engineered DNA Holliday junction (HJ) as a protein-DNA interaction reporter in single-molecule fluorescence resonance energy transfer measurements. By analyzing the single-molecule structural dynamics of the engineered HJ in the presence of various concentrations of both apo- and holo-CueR, we show how CueR interacts with the two conformers of the engineered HJ, forming variable protein-DNA complexes at different protein concentrations and changing the HJ structures. We also show how apo- and holo-CueR differ in their interactions with DNA, and discuss their similarities and differences with other MerR-family metalloregulators. The surprising finding that holo-CueR binds more strongly to DNA than to apo-CueR suggests functional differences among MerR-family metalloregulators, in particular in their mechanisms of switching off gene transcription after activation. The study also corroborates the general applicability of engineered HJs as single-molecule reporters for protein-DNA interactions, which are fundamental processes in gene replication, transcription, recombination, and regulation.
authors: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
authors: Nesha May
authors: NM
authors: Andoy
authors: Susanta K
authors: SK
authors: Sarkar
authors: Qi
authors: Q
authors: Wang
authors: Debashis
authors: D
authors: Panda
authors: Jaime J
authors: JJ
authors: Benítez
authors: Aleksandr
authors: A
authors: Kalininskiy
authors: Peng
authors: P
authors: Chen
mesh_terms: D001483:Base Sequence; D045566:DNA, Cruciform / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015534:Trans-Activators / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D045566:DNA, Cruciform; D029968:Escherichia coli Proteins; D015534:Trans-Activators; C489822:cueR protein, E coli