Single-molecule study of metalloregulator CueR-DNA interactions using engineered Holliday junctions.
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.
Andoy
Nesha May
NM
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.
Sarkar
Susanta K
SK
Wang
Qi
Q
Panda
Debashis
D
Benítez
Jaime J
JJ
Kalininskiy
Aleksandr
A
Chen
Peng
P
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
D045566:DNA, Cruciform; D029968:Escherichia coli Proteins; D015534:Trans-Activators; C489822:cueR protein, E coli