In situ distinction between steroid receptor binding and transactivation at a target gene

Donald P. McDonnell, Zafar Nawaz, Bert W. O'Malley

Research output: Contribution to journalArticle

80 Citations (Scopus)

Abstract

We have developed a DNA interference assay in the yeast Saccharomyces cerevisiae that is designed to indicate the intracellular DNA-binding status of the estrogen receptor. The assay utilizes a promoter containing multiple copies of a GAL4-estrogen receptor binding sequence. This element is designed so that either an estrogen receptor or a GAL4 molecule, but not both, can occupy it simultaneously. The assay is extremely sensitive, and at concentrations of estrogen receptor below that required for maximal transcriptional activation of its target estrogen response element, a quantitative inhibition of GAL4-mediated transcription is seen. Inhibition occurs though the disruption of complex cooperative interactions among the GAL4 molecules in this reporter. The data obtained from our experiments show that at low concentrations of receptor, hormone is required to promote DNA binding. Overexpression of receptor leads to occupation of the estrogen receptor element in the absence of ligand. In contrast, this latter receptor form will not activate transcription. Our results are consistent with a two-step process for receptor activation. Ligand first causes dissociation of receptor from an inhibitory complex within the cell and produces a DNA-binding form. Second, it converts receptor to a transcriptionally competent form. With use of this yeast model system, these two steps can be distinguished in situ.

Original languageEnglish
Pages (from-to)4350-4355
Number of pages6
JournalMolecular and Cellular Biology
Volume11
Issue number9
StatePublished - Dec 1 1991
Externally publishedYes

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Steroid Receptors
Estrogen Receptors
Transcriptional Activation
Genes
DNA
Yeasts
Ligands
Response Elements
Occupations
Saccharomyces cerevisiae
Estrogens
Hormones

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

In situ distinction between steroid receptor binding and transactivation at a target gene. / McDonnell, Donald P.; Nawaz, Zafar; O'Malley, Bert W.

In: Molecular and Cellular Biology, Vol. 11, No. 9, 01.12.1991, p. 4350-4355.

Research output: Contribution to journalArticle

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