Hirling H, Emery-Goodman A, Thompson N, Neupert B, Seiser C, Kühn L C
Swiss Institute for Experimental Cancer Research, Genetics Unit, Epalinges.
Nucleic Acids Res. 1992 Jan 11;20(1):33-9. doi: 10.1093/nar/20.1.33.
Iron regulatory factor (IRF), also called iron responsive element-binding protein (IRE-BP), is a cytoplasmic RNA-binding protein which regulates post-transcriptionally transferrin receptor mRNA stability and ferritin mRNA translation. By using the polymerase chain reaction (PCR) and the sequence published by Rouault et al. (1990) a probe was derived which permitted the isolation of three human IRF cDNA clones. Hybridization to genomic DNA and mRNA, as well as sequencing data indicated a single copy gene of about 40 kb specifying a 4.0 kb mRNA that translates into a protein of 98,400 dalton. By in vitro transcription of a assembled IRF cDNA coupled to in vitro translation in a wheat germ extract, we obtained full sized IRF that bound specifically to a human ferritin IRE. In vitro translated IRF retained sensitivity to sulfhydryl oxidation by diamide and could be reactivated by beta-mercaptoethanol in the same way as native placental IRF. An IRF deletion mutant shortened by 132 amino acids at the COOH-terminus was no longer able to bind to an IRE, indicating that this region of the protein plays a role in RNA recognition. Placental IRF has previously been shown to migrate as a doublet on SDS-polyacrylamide gels. After V8 protease digestion the heterogeneity was located in a 65/70 kDa NH2-terminal doublet. The liberated 31 kDa COOH-terminal polypeptide was found to be homogeneous by amino acid sequencing supporting the conclusion of a single IRF gene.
铁调节因子（IRF），也称为铁反应元件结合蛋白（IRE - BP），是一种细胞质RNA结合蛋白，它在转录后水平调节转铁蛋白受体mRNA的稳定性和铁蛋白mRNA的翻译。利用聚合酶链反应（PCR）和鲁奥尔特等人（1990年）发表的序列，获得了一个探针，该探针可用于分离三个人类IRF cDNA克隆。与基因组DNA和mRNA的杂交以及测序数据表明，该单拷贝基因约为40 kb，编码一个4.0 kb的mRNA，翻译成一种98,400道尔顿的蛋白质。通过将组装好的IRF cDNA进行体外转录，并在小麦胚芽提取物中进行体外翻译，我们获得了能特异性结合人铁蛋白IRE的全长IRF。体外翻译的IRF对二酰胺引起的巯基氧化仍保持敏感性，并且可以像天然胎盘IRF一样被β - 巯基乙醇重新激活。一个在COOH末端缩短了132个氨基酸的IRF缺失突变体不再能够与IRE结合，这表明该蛋白质的这一区域在RNA识别中起作用。胎盘IRF先前已被证明在SDS - 聚丙烯酰胺凝胶上迁移时呈双峰状。经V8蛋白酶消化后，异质性位于一个65/70 kDa的NH2末端双峰中。通过氨基酸测序发现释放出的31 kDa COOH末端多肽是均一的，这支持了单一IRF基因的结论。
