The dissociation rate of estrogen receptor alpha from the consensus estrogen response element.
The rate of dissociation of recombinant, purified human estrogen receptor alpha (ERalpha) from a fluorescein-labeled DNA containing the consensus vitellogenin ERE sequence (F-vitERE) was determined in real time using fluorescence anisotropy. The complex of estradiol-occupied ERalpha with F-vitERE had an apparent dissociation rate of 1.48+/-0.06x10(-2) s(-1) and a half-life of 46.6 s at room temperature. The dissociation rate was characterized by a single exponential decay, suggesting that ER dissociates from the DNA as a preformed dimer, rather than as two individual monomers. The association rate of estradiol-occupied ERalpha for the F-vitERE was calculated as 7x10(6) M(-1) s(-1) based on the dissociation rate measured and previous determinations of the equilibrium dissociation constant (Kd) in similar assay conditions (Ozers et al., 1997). In buffer containing various concentrations of salt, the rate of dissociation of estradiol-occupied ERalpha from F-vitERE was accelerated by increasing salt concentrations. Compared to estradiol-occupied ERalpha, the rate of dissociation of unoccupied ERalpha from the F-vitERE was very similar, indicating that estradiol occupancy does not affect the dissociation rate of ERalpha from the ERE.
Szatkowski Ozers
M
M
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA. maryo@panvera.com
Hill
J J
JJ
Ervin
K
K
Royer
C A
CA
Gorski
J
J
D001667:Binding, Competitive; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000235:genetics; D019793:Fluorescein; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011189:Potassium Chloride / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D014819:Vitellogenins / Q000235:genetics
D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D004967:Estrogens; D011960:Receptors, Estrogen; D014819:Vitellogenins; D004958:Estradiol; D011189:Potassium Chloride; D019793:Fluorescein