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 Ele

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• 作者：Yaohuang Ke ; Hanna Sierzputowska-Gracz ; Zofia Gdaniec ; and Elizabeth C. Theil
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：May 23, 2000
• 年：2000
• 卷：39
• 期：20
• 页码：6235 - 6242
• 全文大小：187K
• 年卷期：v.39,no.20(May 23, 2000)
• ISSN：1520-4995

文摘

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 (T_m 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 (T_m increased) and theHL mutant was less helical near the IL/B (ribonuclease T1 sensitive) and less stable (T_m 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.