Mutational analysis of the interaction between ecdysteroid receptor and its response element

The interaction between the partially purified ecdysteroid receptor (EcR) and the mutated ecdysteroid-response element (EcRE) from the hsp27 gene promoter was studied using the gel retardation competition assay. The results suggest that the EcR-hsp27 EcRE contact sites are made predominantly by base pairs which are at positions −7, −6, −5, −2, −1 and +2, +5, +6 of the hsp27 EcRE palindrome. An increase or decrease on the spacing between the half-palindromes reduces the affinity of the hsp27 EcRE to the receptor, while a mutation of the central A/T base pair to C/G has practically no effect on EcR binding. Unlike the glucocorticoid-response element and the estrogen-response element, the base pairs placed at positions −3, −4 and +1, +3, +4 of the hsp27 EcRE palindrome can be mutated without effect on the EcR binding.