Internal loop/bulge and hairpin loop of the iron-responsive element of ferritin mRNA contribute to maximal iron regulatory protein 2 binding and translational regulation in the iso-iron-responsive element/iso-iron regulatoryprotein family

Iron-responsive elements (IREs), a natural group of mRNA-specific sequences , bind iron regulatory proteins (IRPs) differentially and fold into hairpin s [with a hexaloop (HL) CAGUGX] with helical distortions: an internal loop/ bulge (IL/B) (UGC/C) or C-bulge. C-bulge iso-IREs bind IRP2 more poorly, as oligomers (n = 28-30), and have a weaker signal response in vivo. Two tran s-loop GC base pairs occur in the ferritin IRE (IL/B and HL) but only one i n C-bulge iso-IREs (HL); metal ions and protons perturb the IL/B [Gdaniec e t al. (1998) Biochemistry 37, 1505-1512]. IRE function (translation) and ph ysical properties (T-m and accessibility to nucleases) are now compared for IL/B and C-bulge IREs and for HL mutants. Conversion of the IL/B into a C- bulge by a single deletion in the IL/B or by substituting the HL CG base pa ir with UA both derepressed ferritin synthesis 4-fold in rabbit reticulocyt e lysates (IRP1 + IRP2), confirming differences in IRP2 binding observed fo r the oligomers. Since the engineered C-bulge IRE was more helical near the IL/B [Cu(phen)(2) resistant] and more stable (T-m increased) and the HL mu tant was less helical near the IL/B (ribonuclease T1 sensitive) and less st able (T-m decreased), both CG trans-loop base pairs contribute to maximum I RP2 binding and translational regulation. The H-1 NMR spectrum of the MS-IR E complex revealed, in contrast to the localized IL/B effects of Co(III) he xaammine observed previously, perturbation of the IL/B plus HL and interloo p helix. The lower stability and greater helix distortion in the ferritin I L/B-IRE compared to the C-bulge iso-IREs create a combinatorial set of RNA/ protein interactions that control protein synthesis rates with a range of s ignal sensitivities.