Phosphate-sensitive binding of the estrogen receptor to its response elements

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The DNA sequence of estrogen response elements (ERE) is known, but the role of electrostatic interactions between the DNA backbone and the estrogen receptor (ER) in binding is unclear.
• The contribution of these backbone contacts to sequence-specific DNA binding has not been previously determined.

Purpose of the Study:

• To investigate the involvement of electrostatic contacts between the ER and the sugar-phosphate backbone of the ERE.
• To determine the contribution of these contacts to sequence-specific DNA binding and binding affinity.

Main Methods:

• Phosphate ethylation interference assays were used to examine ER interactions with ERE phosphate residues.
• Electrophoretic gel mobility shift assays (EMSA) were employed to analyze specific ER-DNA complexes.

Main Results:

• Phosphate ethylation interference revealed essential phosphate contacts for specific ER binding to a perfect ERE, with a 5' stagger, indicating ER dimer binding in the DNA major groove.
• Imperfect EREs showed reduced interactions with the ER backbone compared to perfect EREs.
• The absence of specific backbone contacts correlated with reduced ER binding affinity for imperfect EREs.

Conclusions:

• Specific electrostatic contacts between the ER and the DNA sugar-phosphate backbone are critical for sequence recognition.
• These backbone interactions contribute significantly to the high-affinity binding of the estrogen receptor to its cognate response elements.