Dix D J, Lin P N, McKenzie A R, Walden W E, Theil E C
Department of Biochemistry, North Carolina State University, Raleigh 27695.
J Mol Biol. 1993 May 20;231(2):230-40. doi: 10.1006/jmbi.1993.1278.
Ferritin and transferrin receptors are co-ordinately regulated by the same RNA-protein interaction: the conserved iron regulatory element (IRE) in mRNA and the IRE-binding protein (IRE-BP/IRP/FRP/P-90). The 28 nucleotide IRE in ferritin mRNA is a single copy, with base-paired flanking regions (FL), located near the 5' cap. In the transferrin receptor mRNA, the IRE is located in the 3' untranslated region, as five variable copies and lacking predicted base-paired flanking regions; an alternate predicted structure without IREs has similar stability. When iron is scarce, ferritin mRNA does not form polyribosomes whereas the transferrin receptor mRNA is translated; when iron is abundant, ferritin mRNA forms polyribosomes and the transferrin receptor mRNA is degraded. To investigate structures which contribute to differences in the regulation of the two mRNAs, the effect of mutation of the ferritin FL was studied. Changes in structure (changes in reactivity with RNase V1 and RNase S1. Fe-bleomycin) and changes in function (translation in rabbit reticulocyte extracts) were compared for mutant and wild-type FL sequences in ferritin mRNA. The disruption of a triplet of base-pairs in the FL had diminished regulation; a second mutation to restore the triplet base-pairs conferred wild-type translational regulation. Conformation of the mutant RNA-IRE-BP complex was also different. We show that the triplet of base-pairs is conserved; the triplet is also the location of IRE-BP-dependent conformational changes in the FL structure previously observed. Increasing FL base-pairs had no effect on function. Structural changes associated with altered function included bleomycin sites in the IRE, suggesting an alternate conformation of the hairpin, and different base-stacking (V1 sensitivity) in the FL. The function of the FL, which is altered by mutation of phylogenetically conserved triplet base-pairs, may be enhancement of formation of a particular IRE stem-loop-protein interaction.
铁蛋白和转铁蛋白受体由相同的RNA-蛋白质相互作用协同调节：mRNA中保守的铁反应元件（IRE）和IRE结合蛋白（IRE-BP/IRP/FRP/P-90）。铁蛋白mRNA中的28个核苷酸IRE是单拷贝的，具有碱基配对的侧翼区域（FL），位于5'帽附近。在转铁蛋白受体mRNA中，IRE位于3'非翻译区，有五个可变拷贝且缺乏预测的碱基配对侧翼区域；一种没有IRE的替代预测结构具有相似的稳定性。当铁缺乏时，铁蛋白mRNA不形成多核糖体，而转铁蛋白受体mRNA被翻译；当铁丰富时，铁蛋白mRNA形成多核糖体，转铁蛋白受体mRNA被降解。为了研究导致两种mRNA调节差异的结构，研究了铁蛋白FL突变的影响。比较了铁蛋白mRNA中突变型和野生型FL序列的结构变化（与RNase V1和RNase S1、铁-博来霉素反应性的变化）和功能变化（兔网织红细胞提取物中的翻译）。FL中一个三联体碱基对的破坏降低了调节作用；恢复三联体碱基对的第二个突变赋予了野生型翻译调节作用。突变RNA-IRE-BP复合物的构象也不同。我们表明三联体碱基对是保守的；该三联体也是先前观察到的FL结构中IRE-BP依赖性构象变化的位置。增加FL碱基对没有功能影响。与功能改变相关的结构变化包括IRE中的博来霉素位点，表明发夹结构的另一种构象，以及FL中不同的碱基堆积（对V1敏感）。FL的功能因系统发育保守的三联体碱基对突变而改变，其功能可能是增强特定IRE茎环-蛋白质相互作用的形成。
