LOCALIZATION OF AN RNA-BINDING ELEMENT OF THE IRON-RESPONSIVE ELEMENT-BINDING PROTEIN WITHIN A PROTEOLYTIC FRAGMENT CONTAINING IRON COORDINATION LIGANDS

The iron responsive element binding protein (IRE-BP) regulates iron st orage and uptake in response to iron. This control results from the in teraction of the IRE-BP with the iron responsive element (IRE), a cons erved sequence/structure element located near the 5' end of all ferrit in mRNAs and in the 3' UTR of transferrin receptor mRNAs. Proteolysis was used to probe for functional elements of the IRE-BP. Partial chymo trypsin digestion generates a simple digestion pattern yielding fragme nts of 68, 56, 41, and 30 kDa. The 68 and 30 kDa fragments are derived from a single cleavage at Trp(623). Further cleavages of the 68 kDa p olypeptide yield the 56 and 41 kDa peptides. A combination of UV-cross linking and chymotrypsin digestion was used to localize an RNA binding element within the C-terminus of the 68 kDa fragment, between amino a cid residues 480 and 623. This region includes cysteine residues 503 a nd 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 element s within the IRE-BP, in addition to those located between residues 480 and 623, are required for high affinity binding to the IRE.