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 regulatory protein family.
Iron-responsive elements (IREs), a natural group of mRNA-specific sequences, bind iron regulatory proteins (IRPs) differentially and fold into hairpins [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 trans-loop GC base pairs occur in the ferritin IRE (IL/B and HL) but only one in C-bulge iso-IREs (HL); metal ions and protons perturb the IL/B [Gdaniec et al. (1998) Biochemistry 37, 1505-1512]. IRE function (translation) and physical 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 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 engineered C-bulge IRE was more helical near the IL/B [Cu(phen)(2) resistant] and more stable (T(m) increased) and the HL mutant was less helical near the IL/B (ribonuclease T1 sensitive) and less stable (T(m) decreased), both CG trans-loop base pairs contribute to maximum IRP2 binding and translational regulation. The (1)H NMR spectrum of the Mg-IRE complex revealed, in contrast to the localized IL/B effects of Co(III) hexaammine observed previously, perturbation of the IL/B plus HL and interloop helix. The lower stability and greater helix distortion in the ferritin IL/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 signal sensitivities.
Ke
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CHORI (Children's Hospital Oakland Research Institute), Oakland, California 94609-1673, USA.
Sierzputowska-Gracz
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H
Gdaniec
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Z
Theil
E C
EC
D000818:Animals; D003300:Copper / Q000737:chemistry; D005293:Ferritins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D006358:Hot Temperature; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D008116:Lizards; D008274:Magnesium / Q000737:chemistry; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D010618:Phenanthrolines / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D011817:Rabbits; D011892:Rana catesbeiana; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology; D006163:Ribonuclease T1 / Q000737:chemistry
D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D010618:Phenanthrolines; D020033:Protein Isoforms; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C014741:bis(1,10-phenanthroline)copper(2+) ion; D003300:Copper; D005293:Ferritins; D006163:Ribonuclease T1; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2; D008274:Magnesium