Leibold E A, Laudano A, Yu Y
Department of Medicine, University of Utah School of Medicine, Salt Lake City 84132.
Nucleic Acids Res. 1990 Apr 11;18(7):1819-24. doi: 10.1093/nar/18.7.1819.
The synthesis of both transferrin receptor (TfR) and ferritin is regulated post-transcriptionally by iron. This is mediated by iron responsive elements (IREs) in the 5'- and 3'-untranslated regions, respectively, of TfR and ferritin mRNAs. Although these IREs have different sequences, they both form a characteristic stem-loop. We used competition assays and partial peptide mapping of UV-crosslinked ferritin and TfR IRE-protein complexes to show that the cytosolic protein binding to the ferritin 5'-IRE, the iron-responsive element binding protein (IRE-BP), also binds to TfR 3'-IREs. To identify the structural requirements necessary for RNA-protein binding, ferritin IRE RNAs were synthesized which contained altered secondary structures and base substitutions. Affinities of these RNAs for IRE-BP were assayed in RNA-protein binding gels. Substitutions disrupting base-pairing of the stem prevented IRE-BP binding. Substitutions which restored base-pairing also restored IRE-BP binding. We conclude that the IRE-BP binds to both ferritin and TfR IREs and recognizes a particular IRE conformation.
转铁蛋白受体（TfR）和铁蛋白的合成均受到铁的转录后调控。这是由TfR和铁蛋白mRNA的5'-和3'-非翻译区中的铁反应元件（IRE）介导的。尽管这些IRE具有不同的序列，但它们都形成一个特征性的茎环结构。我们使用竞争试验以及对紫外线交联的铁蛋白和TfR IRE-蛋白质复合物进行部分肽图谱分析，以表明与铁蛋白5'-IRE结合的胞质蛋白，即铁反应元件结合蛋白（IRE-BP），也与TfR 3'-IRE结合。为了确定RNA-蛋白质结合所需的结构要求，合成了含有改变的二级结构和碱基取代的铁蛋白IRE RNA。在RNA-蛋白质结合凝胶中测定这些RNA对IRE-BP的亲和力。破坏茎碱基配对的取代阻止了IRE-BP的结合。恢复碱基配对的取代也恢复了IRE-BP的结合。我们得出结论，IRE-BP与铁蛋白和TfR IRE均结合，并识别特定的IRE构象。
