Metalloregulator CueR biases RNA polymerase's kinetic sampling of dead-end or open complex to repress or activate transcription.
Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ(70)-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator-DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)--a Cu(+)-responsive MerR-family metalloregulator--modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR-PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853;
Danya J
DJ
Martell
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853;
Chandra P
CP
Joshi
Department of Microbiology, Cornell University, Ithaca, NY 14853.
Ahmed
A
Gaballa
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853;
Ace George
AG
Santiago
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853;
Tai-Yen
TY
Chen
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853;
Won
W
Jung
Department of Microbiology, Cornell University, Ithaca, NY 14853.
John D
JD
Helmann
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853; pc252@cornell.edu.
Peng
P
Chen
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000465:Algorithms; D001483:Base Sequence; D002232:Carbocyanines / Q000737:chemistry; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D002232:Carbocyanines; D027682:Cation Transport Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012808:Sigma Factor; D015534:Trans-Activators; C489822:cueR protein, E coli; C089187:cyanine dye 3; C085321:cyanine dye 5; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases
MerR-family regulators; metal-responsive transcription regulation; protein–DNA interaction dynamics; single-molecule FRET