title: Interplay of flexibility and stability in the control of estrogen receptor activity.
abstract: Previously, we have identified an imperfect estrogen response element (rtERE) in the promoter of the rainbow trout vitellogenin gene. Although this ERE leads to a lower transcriptional activation, a better estradiol stimulation in vivo as compared to consensus ERE (EREcs) was observed. Here we examine the ability of recombinant human estrogen receptor alpha (rhERalpha) to bind DNA containing the EREcs or the natural imperfect rtERE, which contains three mismatches. At low salt concentration, whatever the ERE sequence, dissociation equilibrium constants of the specific rhERalpha-ERE complexes are similar (K(D) = 2 nM) with the same stoichiometry. As salt concentration increases from 80 to 200 mM KCl, the affinity of the rhERalpha-rtERE complex largely diminishes whereas that of rhERalpha-EREcs seems less affected. Hence the nature of the interactions stabilizing these complexes is different: more ionic in rhERalpha-rtERE as compared to rhERalpha-EREcs. Moreover, kinetic measurements showed that specific rhERalpha-ERE complexes exhibit shorter half-lives (few seconds) and that the rhERalpha-EREcs complex is more stable (33 s) than the complex that formed with rtERE (19.8 s), in accordance with equilibrium binding results. Finally, dynamic studies of rhERalpha have shown that the protein fluctuations are damped when the salt concentration increases or when bound to ERE and all the more with rtERE. The interplay of affinity, complex half-lives, and protein dynamics in the transcriptional regulation of estrogen receptor is discussed.
authors.affiliation: Laboratoire des Polymères, Propriétés aux Interfaces et Composites, Université de Bretagne-Sud, CER Yves Coppens, Campus de Tohannic, BP573, 56017 Vannes CEDEX, France.
authors.forename: A
authors.initials: A
authors.lastname: Bouter
authors.forename: V
authors.initials: V
authors.lastname: Le Tilly
authors.forename: O
authors.initials: O
authors.lastname: Sire
mesh_terms: D000818:Animals; D020137:Base Pair Mismatch; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D012492:Salts / Q000737:chemistry; D013816:Thermodynamics; D015533:Transcriptional Activation; D014364:Tryptophan / Q000737:chemistry
chemical_list: D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D008024:Ligands; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012492:Salts; D014364:Tryptophan