Steroid hormones are believed to influence gene transcription in target tissues at 5 feet-pre-gene DNA sequences of specific genes via a transformed cytosolic receptor. Cytosolic allosteric activation by the steroid imparts to the receptor a conformation having nuclear affinity and both specific and non-specific DNA binding capacities. This model is appealing in its simplicity, but contains no specific mechanism for """"""""proofreading"""""""" the identify of the inducing compound. Such proofreading may have importance, e.g. in light of the known ability of various steroids to cross-react """"""""incorrectly"""""""" with receptor proteins (e.g. androgens with estrogen receptors). We propose to isolated estradiol 17 beta responsive rat uterine genes for determination of the estrogen receptor's capacity to proofread inducing signals by comparison of signal structure to cavities in B-DNA at or near receptor binding sequences. Such a comparative process might most simply proceed by direct intercalation of the steroid into B-DNA, a concept supported by a number of biophysical and modeling studies. A collection of cDNA clones representing early induced or suppressed transcripts will be prepared from whole estradiol-induced or non-induced uterine poly A-mRNA populations. This collections will provide probes for isolation of sequences corresponding to responsive genes from a rat genomic library contained on phage. Partial chemical sequencing of appropriate isolated DNA fragments in conjunction with estrogen receptor protein protection of these sequences from DNase I treatment will provide a pre-gene receptor binding sequence and, in all likelihood, a consensus sequence for such binding from comparision of different pregene sequences. The ability of estrogen receptor to affect an enhanced binding of estradiol or diethylstilbestrol of these estrogens to pre-gene DNA sequences will be explored by screening for enhanced nicking at sites of estrogen-DNA complex formation and/or chemical analysis of estrogen-nuceleoside complexes formed in vitro. In addition, the ability of isolated pre-gene DNA sequences to interact specifically with steroids in the absence of receptor wll be assessed by the effects of steroids on gel electrophoretic mobility of supercoiled DNA. This project is designed to maximize useful information and collaborative interactions at each step while developing tools for disclosing unexplored stereochemical principles for intracellular steroid perception. The tools derived will also have a definite applicability for in vivo verification of the chemical principles derived in vitro.
| Hendry, L B; Bransome Jr, E D; Lehner, A F et al. (1986) The stereochemical complementarity of DNA and reproductive steroid hormones correlates with biological activity. J Steroid Biochem 24:843-52 |
