Identification of the glucocorticoid receptor binding site at the 5'-flanking region of mouse metallothionein I gene: the effect of base substitutions on binding efficiency]

Interaction of highly purified glucocorticoid receptor complex (GIRC) with synthetic DNA-fragment of mouse metallotionein 1 gene promoter from -209 to -252 b.p. (MTwt) was investigated. By means of nitrocellulose filter binding assay this fragment was shown to contain specific GIRC-binding site. In order to analyse the fine structure of the site, two variants of this DNA-fragment were synthesized and used in gel retardation assay. GIRC specific binding was shown to retain throughout interaction with the fragment in which all base pairs in the surroundings of generally accepted GIRC-binding site consensus G--ACA---TGTTCT C--TGT---ACAAGA were substituted by means of transitions, but it was weaker than the GIRC-binding with MTwt, where the mentioned consensus was situated in the natural surroundings. Complete loss of the GIRC-binding ability was observed when five CG pairs were substituted by AT ones. Two of the CG pairs belonged to the mentioned consensus. Comparison of the data obtained with results of computer analysis allows to consider the consensus as a "core" of GIRC-binding site, flanked with additional elements, interacting with GIRC.