Contacts Between Steroid Hormone Receptors and Thymines in DNA: An Interference Method

Understanding the mechanisms by which regulatory proteins recognize genetic information stored in DNA relies on the availability of methods to analyze their interaction with individual nucleotides and their reactive groups. Here we describe the use of KMnO4to analyze the contacts between steroid hor...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1990-09, Vol.87 (18), p.7180-7184
Main Authors: Truss, Mathias, Chalepakis, Georges, Beato, Miguel
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Binding Sites
Biological and medical sciences
Cell receptors
Cell structures and functions
Chemical bases
DNA
DNA - metabolism
Fundamental and applied biological sciences. Psychology
Gels
Hormone receptors
Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors
Hydrazines
Mammary Tumor Virus, Mouse - genetics
Molecular and cellular biology
Molecular Sequence Data
Oligodeoxyribonucleotides - chemical synthesis
Oligodeoxyribonucleotides - metabolism
Oligonucleotides
Palindromes
Plasmids
Potassium Permanganate
Receptors
Receptors, Steroid - metabolism
Restriction Mapping
Thymine
Transfection
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Summary:Understanding the mechanisms by which regulatory proteins recognize genetic information stored in DNA relies on the availability of methods to analyze their interaction with individual nucleotides and their reactive groups. Here we describe the use of KMnO4to analyze the contacts between steroid hormone receptors and thymines within a hormone responsive element. Although several pyrimidine residues are highly conserved among different receptor binding sites their participation in sequence recognition has not been directly studied. Using an interference procedure based on selective modification of the thymine ring by KMnO4, we detect intimate contacts between the glucocorticoid or progesterone receptors and three thymine residues within the promoter distal receptor binding site of the mouse mammary tumor virus (-190/-160). A comparison of binding data obtained with oligonucleotides containing desoxyuridine, bromodesoxyuridine, cytosine, or 5'-methylcytosine instead of thymines demonstrates that the methyl group of those three thymines contributes to the free energy of binding. This simple method could be of general utility for the study of sequence-specific protein-DNA interactions.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.87.18.7180