| Abstract
| - Iron-responsive elements (IREs), a natural group of mRNA-specific sequences, bind ironregulatory proteins (IRPs) differentially and fold into hairpins [with a hexaloop (HL) CAGUGX] withhelical distortions: an internal loop/bulge (IL/B) (UGC/C) or C-bulge. C-bulge iso-IREs bind IRP2 morepoorly, as oligomers (n = 28−30), and have a weaker signal response in vivo. Two trans-loop GC basepairs occur in the ferritin IRE (IL/B and HL) but only one in C-bulge iso-IREs (HL); metal ions andprotons perturb the IL/B [Gdaniec et al. (1998) Biochemistry 37, 1505−1512]. IRE function (translation)and physical properties (Tm and accessibility to nucleases) are now compared for IL/B and C-bulge IREsand for HL mutants. Conversion of the IL/B into a C-bulge by a single deletion in the IL/B or by substitutingthe HL CG base pair with UA both derepressed ferritin synthesis 4-fold in rabbit reticulocyte lysates(IRP1 + IRP2), confirming differences in IRP2 binding observed for the oligomers. Since the engineeredC-bulge IRE was more helical near the IL/B [Cu(phen)2 resistant] and more stable (Tm increased) and theHL mutant was less helical near the IL/B (ribonuclease T1 sensitive) and less stable (Tm decreased), bothCG trans-loop base pairs contribute to maximum IRP2 binding and translational regulation. The 1H NMRspectrum of the Mg−IRE complex revealed, in contrast to the localized IL/B effects of Co(III) hexaammineobserved previously, perturbation of the IL/B plus HL and interloop helix. The lower stability and greaterhelix distortion in the ferritin IL/B-IRE compared to the C-bulge iso-IREs create a combinatorial set ofRNA/protein interactions that control protein synthesis rates with a range of signal sensitivities.
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