Eisenstein R S, Barton H A, Pettingell W H, Bomford A B
Department of Nutritional Sciences, University of Wisconsin, Madison 53706, USA.
Arch Biochem Biophys. 1997 Jul 1;343(1):81-91. doi: 10.1006/abbi.1997.0144.
Ferritin mRNAs are translationally regulated by the binding of either of two cytosolic proteins, iron regulatory protein 1 (IRP1) or IRP2, to the iron responsive element (IRE) located in their 5' untranslated region (UTR). Rat liver IRP1 was purified by anion exchange, gel filtration, and affinity chromatography using a concatemerized version of the IRE. Two bands with M(r) of 95,000 and 100,000 were observed by reducing SDS-PAGE. A single protein was responsible for both bands since: (1) [32P]IRE RNA specifically cross-linked to both components; (2) alkylation with iodoacetamide resulted in formation of a single species with M(r) of 95,000; and (3) they possessed identical peptide patterns after digestion with cyanogen bromide. The N-terminal sequence of rat liver IRP1 was MKNPFAHLAEPLDPAQPGKKFNLNKLEDSRYGRLPFXIRVLLEAAV which is identical to the sequence deduced from the cDNA. Rat liver IRP1 has an amino acid composition similar to that of bovine liver caconitase. Several species of IRP1 were observed by two-dimensional gel electrophoresis with pIs ranging from 7.5 to 8.0. Rat liver IRP1 bound the IRE with high affinity (K(D) = 0.04 nM) and repressed translation of ferritin mRNA in vitro. IRP1 bound 100-fold less well to an IRE variant and failed to significantly repress translation of a ferritin mRNA containing the mutated IRE. We conclude that decreases in the affinity of interaction between IRP1 and the IRE, of a magnitude similar to that observed when the binding protein in converted to c-aconitase, are sufficient to significantly enhance translation of ferritin mRNA in vitro.
铁蛋白mRNA的翻译受两种胞质蛋白（铁调节蛋白1，即IRP1或铁调节蛋白2，即IRP2）之一与位于其5'非翻译区（UTR）的铁反应元件（IRE）结合的调控。大鼠肝脏IRP1通过阴离子交换、凝胶过滤和使用IRE串联体的亲和色谱法进行纯化。通过还原SDS-PAGE观察到两条分子量分别为95,000和100,000的条带。这两条带由单一蛋白质产生，原因如下：（1）[32P]IRE RNA与两种组分均发生特异性交联；（2）用碘乙酰胺烷基化导致形成单一的分子量为95,000的物种；（3）用溴化氰消化后它们具有相同的肽图谱。大鼠肝脏IRP1的N端序列为MKNPFAHLAEPLDPAQPGKKFNLNKLEDSRYGRLPFXIRVLLEAAV，与从cDNA推导的序列相同。大鼠肝脏IRP1的氨基酸组成与牛肝脏顺乌头酸酶相似。通过二维凝胶电泳观察到几种IRP1，其等电点范围为7.5至8.0。大鼠肝脏IRP1以高亲和力（K(D)=0.04 nM）结合IRE，并在体外抑制铁蛋白mRNA的翻译。IRP1与IRE变体的结合能力低100倍，并且不能显著抑制含有突变IRE的铁蛋白mRNA的翻译。我们得出结论，IRP1与IRE之间相互作用亲和力的降低幅度与结合蛋白转化为顺乌头酸酶时观察到的幅度相似，足以在体外显著增强铁蛋白mRNA的翻译。
