Iron Regulatory Element and Internal Loop/Bulge Structure for Ferritin mRNA Studied by Cobalt(III) Hexammine Binding, Molecular Modeling, and NMR Spectroscopy

Abstract

The ferritin IRE, a highly conserved (96−99% in vertebrates) mRNA translation regulatory element in animal mRNA, was studied by molecular modeling (using MC-SYM and DOCKING) and by NMR spectroscopy. Cobalt(III) hexammine was used to model hydrated Mg²⁺. IRE isoforms in other mRNAs regulate mRNA translation or stability; all IREs bind IRPs (iron regulatory proteins). A G·C base pair, conserved in ferritin IREs, spans an internal loop/bulge in the middle of an A-helix and, combined with a dynamic G·U base pair, formed a pocket suitable for Co(III) hexammine binding. On the basis of the effects of Co(III) hexammine on the ¹H NMR spectrum and results of automatic docking into the IRE model, the IRE bound Co(III) hexammine at the pocket in the major groove; Mg²⁺ may bind to the IRE at the same site on the basis of an analogy to Co(III) hexammine and on the Mg²⁺ inhibition of Cu(phen)₂ cleavage at the site. Distortion of the IRE helix by the internal loop/bulge near a conserved unpaired C required for IRP binding and adjacent to an IRP cross-linking site suggests a role for the pocket in ferritin IRE/IRP interactions.