Multiple determinants within iron-responsive elements dictate iron regulatory protein binding and regulatory hierarchy.
Iron regulatory proteins (IRPs) are iron-regulated RNA binding proteins that, along with iron-responsive elements (IREs), control the translation of a diverse set of mRNA with 5' IRE. Dysregulation of IRP action causes disease with etiology that may reflect differential control of IRE-containing mRNA. IREs are defined by a conserved stem-loop structure including a midstem bulge at C8 and a terminal CAGUGH sequence that forms an AGU pseudo-triloop and N19 bulge. C8 and the pseudo-triloop nucleotides make the majority of the 22 identified bonds with IRP1. We show that IRP1 binds 5' IREs in a hierarchy extending over a ninefold range of affinities that encompasses changes in IRE binding affinity observed with human L-ferritin IRE mutants. The limits of this IRE binding hierarchy are predicted to arise due to small differences in binding energy (e.g., equivalent to one H-bond). We demonstrate that multiple regions of the IRE stem not predicted to contact IRP1 help establish the binding hierarchy with the sequence and structure of the C8 region displaying a major role. In contrast, base-pairing and stacking in the upper stem region proximal to the terminal loop had a minor role. Unexpectedly, an N20 bulge compensated for the lack of an N19 bulge, suggesting the existence of novel IREs. Taken together, we suggest that a regulatory binding hierarchy is established through the impact of the IRE stem on the strength, not the number, of bonds between C8 or pseudo-triloop nucleotides and IRP1 or through their impact on an induced fit mechanism of binding.
Goforth
Jeremy B
JB
Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
Anderson
Sheila A
SA
Nizzi
Christopher P
CP
Eisenstein
Richard S
RS
D000818:Animals; D001052:Apoferritins / Q000378:metabolism; D001483:Base Sequence / Q000502:physiology; D006801:Humans; D035941:Iron Regulatory Protein 1 / Q000378:metabolism; D035925:Iron-Regulatory Proteins / Q000378:metabolism*; D053858:Metabolic Networks and Pathways / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism; D011817:Rabbits; D020218:Response Elements / Q000235:genetics* / Q000502:physiology*; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity / Q000235:genetics
D035925:Iron-Regulatory Proteins; D016601:RNA-Binding Proteins; D001052:Apoferritins; D035941:Iron Regulatory Protein 1