Related Experiment Video
Updated: Aug 9, 2026

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example
Published on: December 4, 2014
Localization of an RNA binding element of the iron responsive element binding protein within a proteolytic fragment
1Department of Microbiology and Immunology, University of Illinois at Chicago 60612.
Abstract:
The iron responsive element binding protein (IRE-BP) regulates iron storage and uptake in response to iron. This control results from the interaction of the IRE-BP with the iron responsive element (IRE), a conserved sequence/structure element located near the 5' end of all ferritin mRNAs and in the 3' UTR of transferrin receptor mRNAs. Proteolysis was used to probe for functional elements of the IRE-BP. Partial chymotrypsin digestion generates a simple digestion pattern yielding fragments of 68, 56, 41, and 30 kDa. The 68 and 30 kDa fragments are derived from a single cleavage at Trp623. Further cleavages of the 68 kDa polypeptide yield the 56 and 41 kDa peptides. A combination of UV-crosslinking and chymotrypsin digestion was used to localize an RNA binding element within the C-terminus of the 68 kDa fragment, between amino acid residues 480 and 623. This region includes cysteine residues 503 and 506 which have been shown to be required for iron-sulfur cluster assembly and for iron regulation of the IRE-BP. Proteolytic fragments of the IRE-BP that contain this RNA binding region can be crosslinked to the IRE but do not bind with high affinity, suggesting that elements within the IRE-BP, in addition to those located between residues 480 and 623, are required for high affinity binding to the IRE.
Related Concept Videos
Conserved Binding Sites
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
Ribosomal RNA Synthesis
Ribosome biogenesis begins with the synthesis of 5S and 45S pre-rRNAs by distinct RNA polymerases. The primary transcripts are extensively processed and modified before they are bound and folded by ribosomal proteins and assembly factors,...
The Early Endosome: Endocytosis of Transferrin
Ribosome Profiling
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...
Directing Proteins to the Rough Endoplasmic Reticulum
Transcriptional Regulation: Riboswitches

