The dissociation rate of estrogen receptor alpha from the consensus estrogen response element

The rate of dissociation of recombinant, purified human estrogen receptor a lpha (ER alpha) from a fluorescein-labeled DNA containing the consensus vit ellogenin ERE sequence (F-vitERE) was determined in real time using fluores cence anisotropy. The complex of estradiol-occupied ER alpha with F-vitERE had an apparent dissociation rate of 1.48 +/- 0.06 x 10(-2) s(-1) and a hal f-life of 46.6 s at room temperature. The dissociation rate was characteriz ed by a single exponential decay, suggesting that ER dissociates from the D NA as a preformed dimer, rather than as two individual monomers. The associ ation rate of estradiol-occupied ER alpha for the F-vitERE was calculated a s 7 x 10(6) M-1 s(-1) based on the dissociation rate measured and previous determinations of the equilibrium dissociation constant (K-d) in similar as say conditions (Ozers et al., 1997). In buffer containing various concentra tions of salt, the rate of dissociation of estradiol-occupied ER alpha from F-vitERE was accelerated by increasing salt concentrations. Compared to es tradiol-occupied ER alpha, the rate of dissociation of unoccupied ER alpha from the F-vitERE was very similar, indicating that estradiol occupancy doe s not affect the dissociation rate of ER alpha from the ERE. (C) 2001 Elsev ier Science Ireland Ltd. All rights reserved.