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Updated: Aug 11, 2026

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example
Published on: December 3, 2014
The hairpin loop but not the bulged C of the iron responsive element is essential for high affinity binding to iron
1Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455-0347, USA.
Abstract:
Vertebrates control intracellular iron concentration principally through the interaction of iron regulatory proteins with mRNAs that contain an iron responsive element, a small hairpin with a bulged C. The hairpin loop and bulged C have previously been assumed to be critical for binding and have been proposed to make direct contact with the iron regulatory proteins. However, we show here that a U or G can be substituted for the bulged C provided that specific nucleotides are also present within internal loops. The K(d), IC(50) and chemical modifications of the iron responsive element variants are similar to the wild-type. Results are more consistent with a role in which the C-bulge functions to orient the hairpin for optimal protein binding rather than to directly contact the protein. Characterization of these novel iron responsive element variants may facilitate the identification of additional mRNAs whose expression is controlled by iron regulatory proteins, as well as provide insight into the nature of a critical RNA-protein interaction.
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