Harrell C M, McKenzie A R, Patino M M, Walden W E, Theil E C
Department of Biochemistry, North Carolina State University, Raleigh 27695-7622.
Proc Natl Acad Sci U S A. 1991 May 15;88(10):4166-70. doi: 10.1073/pnas.88.10.4166.
The ferritin iron regulatory element (IRE), a conserved sequence of 28 nucleotides in a hairpin loop, is a conserved mRNA-specific translational regulatory element; flanking the IRE are regions of varying sequence, which form 9-17 base pairs close to the 5' cap. P-90 is a ferritin mRNA-specific translation regulatory protein purified from animal liver and reticulocytes. To study the P-90-RNA interaction, protein nucleases (RNase S1 and T1) and chemical nucleases FeEDTA and/or 1,10-phenanthroline-Cu were used as probes of an oligonucleotide (n = 55), containing the IRE and flanking regions (FL), and natural ferritin mRNA. Footprints and "toeprints" showed that P-90 binding was confined to the stem and loop of the IRE itself. However, P-90 altered the structure of the flanking region by increasing base stacking or helicity (RNase V1 sensitivity). Comparison of the reactivity of the IRE and flanking regions in natural mRNA and the 55-mer showed that long-range interactions included protecting bulges, single-stranded, and stacked regions from protein nucleases as well as stabilizing the P-90-RNA interaction. Structural integration of the IRE with the base-paired flanking regions was indicated by common features of reactivity (periodic hypersensitivity to FeEDTA) and changes in the FL region caused by P-90. The increased secondary structure of the IRE flanking regions caused by P-90 binding to the IRE provides a likely mechanism for blocking initiation of ferritin mRNA translation, since the combined structure (IRE + FL) is so close (8-17 nucleotides) to the cap.
铁蛋白铁调节元件（IRE）是发夹环中一段由28个核苷酸组成的保守序列，是一种保守的mRNA特异性翻译调节元件；IRE两侧是序列各异的区域，这些区域在靠近5'帽的位置形成9 - 17个碱基对。P - 90是一种从动物肝脏和网织红细胞中纯化出来的铁蛋白mRNA特异性翻译调节蛋白。为了研究P - 90与RNA的相互作用，蛋白核酸酶（RNase S1和T1）以及化学核酸酶FeEDTA和/或1,10 - 菲咯啉 - 铜被用作一种寡核苷酸（n = 55）的探针，该寡核苷酸包含IRE及其侧翼区域（FL）以及天然铁蛋白mRNA。足迹和“ toe印”显示P - 90的结合局限于IRE自身的茎环结构。然而，P - 90通过增加碱基堆积或螺旋度（对RNase V1敏感）改变了侧翼区域的结构。对天然mRNA和55聚体中IRE及其侧翼区域反应性的比较表明，长程相互作用包括保护凸起、单链和堆积区域免受蛋白核酸酶的作用以及稳定P - 90 - RNA相互作用。IRE与碱基配对的侧翼区域的结构整合通过反应性的共同特征（对FeEDTA的周期性超敏反应）以及P - 90引起的FL区域变化得以体现。P - 90与IRE结合导致IRE侧翼区域二级结构增加，这为阻断铁蛋白mRNA翻译起始提供了一种可能的机制，因为组合结构（IRE + FL）距离帽非常近（8 - 17个核苷酸）。
