Swenson G R, Patino M M, Beck M M, Gaffield L, Walden W E
Department of Microbiology and Immunology, University of Illinois, Chicago 60612.
Biol Met. 1991;4(1):48-55. doi: 10.1007/BF01135557.
The iron-responsive regulation of ferritin mRNA translation is mediated by the specific interaction of the ferritin repressor protein (FRP) with the iron-responsive element (IRE), a highly conserved 28-nucleotide sequence located in the 5' untranslated region of ferritin mRNAs. The IRE alone is necessary and sufficient to confer repression of translation by FRP upon a heterologous message, chloramphenicol acetyltransferase, in an in vitro translation system. The activity of FRP is sensitive to iron in vivo. Cytoplasmic extracts of rabbit kidney cells show reduction of FRP activity when grown in the presence of iron, as detected by RNA band shift assay. Using a nitrocellulose filter binding assay to examine the interaction of FRP with the IRE in more detail, we find that purified FRP has a single high-affinity binding site for the IRE with a Kd of 20-50 pM. Hemin pretreatment decreases the total amount of FRP which can bind to the IRE. This effect is dependent on hemin concentration. Interestingly, the FRP which remains active at a given hemin concentration binds to the IRE with the same high affinity as untreated FRP. A variety of hemin concentrations were examined for their effect on preformed FRP/IRE complexes. All hemin concentrations tested resulted in rapid complex breakdown. The final amount of complex breakdown corresponds to the concentration of hemin present in the reaction. The effect of hemin on FRP activity suggests that a specific hemin binding site exists on FRP.
铁蛋白mRNA翻译的铁响应调节是由铁蛋白阻遏蛋白（FRP）与铁响应元件（IRE）的特异性相互作用介导的，IRE是位于铁蛋白mRNA 5'非翻译区的高度保守的28个核苷酸序列。在体外翻译系统中，单独的IRE对于将FRP介导的翻译抑制赋予异源信息氯霉素乙酰转移酶是必要且充分的。FRP的活性在体内对铁敏感。通过RNA带移分析检测，兔肾细胞的细胞质提取物在铁存在下生长时显示FRP活性降低。使用硝酸纤维素滤膜结合分析更详细地检查FRP与IRE的相互作用，我们发现纯化的FRP对IRE有一个单一的高亲和力结合位点，Kd为20 - 50 pM。血红素预处理降低了可与IRE结合的FRP总量。这种效应取决于血红素浓度。有趣的是，在给定血红素浓度下仍保持活性的FRP与IRE结合的亲和力与未处理的FRP相同。研究了各种血红素浓度对预先形成的FRP/IRE复合物的影响。所有测试的血红素浓度都导致复合物迅速分解。复合物分解的最终量与反应中存在的血红素浓度相对应。血红素对FRP活性的影响表明FRP上存在一个特定的血红素结合位点。
