Characterization of DNA binding and retinoic acid binding properties of retinoic acid receptor

N. A. Yang, Roland Schüle, David J. Mangelsdorf, Ronald M. Evans

Research output: Contribution to journalArticle

106 Citations (Scopus)

Abstract

High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a Kd of 2.1 × H10-10 M.

Original languageEnglish (US)
Pages (from-to)3559-3563
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume88
Issue number9
StatePublished - 1991

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Retinoic Acid Receptors
Tretinoin
DNA
Proteins
Bacterial Proteins
Charcoal
Response Elements
Eukaryotic Cells
Electrophoretic Mobility Shift Assay
Cell Extracts
Hot Temperature
Gels
Hormones
Escherichia coli
Amino Acids

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

Characterization of DNA binding and retinoic acid binding properties of retinoic acid receptor. / Yang, N. A.; Schüle, Roland; Mangelsdorf, David J.; Evans, Ronald M.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 88, No. 9, 1991, p. 3559-3563.

Research output: Contribution to journalArticle

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AB - High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a Kd of 2.1 × H10-10 M.

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