IRON REGULATION OF GENE EXPRESSION

铁是一种必不可少的元素，是大多数细胞生存和生存所必需的 成长。过量的铁对细胞是有毒的。因此，它在细胞中的水平 受到严格监管。哺乳动物细胞维持稳定的胞浆 通过调节它们对铁的摄取来提高游离铁的浓度 转铁蛋白受体(TFR)和通过将细胞内的铁隔离到 铁蛋白。铁蛋白的合成是由铁诱导的，其机制是 将潜伏的铁蛋白mRNA转变为主动翻译多聚体。TFR 铁减少了合成，促进了TFR mRNA的失稳。 铁对铁蛋白和转铁蛋白受体合成的协调调节 由铁响应元素或IRES控制。这些IRES位于 铁蛋白和转铁蛋白受体mRNAs的5‘和3’非翻译区， 分别进行了分析。IRES形成特征茎环结构并结合 胞浆蛋白，铁反应蛋白或IRE-BPS。两个这样的人 对IRE-BP B1和IRE-BP B2蛋白进行了部分鉴定。这个 拟议的实验旨在确定通过什么机制 铁蛋白和转铁蛋白受体mRNAs受IRE-Bps的协调调节。 第一个特定目的是通过筛选一只大鼠来分离IRE-BP基因 含氨基酸衍生寡核苷酸的肝lambda基因文库 IRE-BP序列，主要为大鼠IRE-BP。该cdna序列将是 确定并将推导出的氨基酸序列与氨基酸进行比较 其他蛋白质的酸序列来识别保守的区域 IRE-BP可能在RNA-蛋白质识别中具有重要的功能。这个 第二个特异性目的是研究IRE-BP B1在大鼠肝癌中的表达 细胞。IRE-BP基因将被用于确定IRE-BP B1的表达 对细胞内铁和/或血红素水平的变化以及对 确定转录或转录后机制是 参与调节IRE-BP B1的合成。第三个具体目标 是否通过检测铁蛋白和转铁蛋白受体IRES的预测次级 含改变核苷酸序列的IRE RNA的合成及检测 这些突变体与IRE-BP B1结合。这些研究将允许 IRE结构与生物学功能的相关性。第四个具体问题 目的是确定大鼠肝脏中的第二种蛋白质IRE-BP的功能 B2.将从大鼠肝脏中分离并测序IRE-BP B2蛋白，并 它的功能和结构中的功能决定因素将是 特色化的。第五个也是最后一个具体目标是确定是否有 铁调节转铁蛋白受体基因表达的其他因素。一个没有手机的地方 将开发系统来识别多倍体或胞质因子 而不是IRE-BPS，这可能特异性地影响TFR mRNA的降解。

Iron is an essential element and is required by most cells for survival and growth. In excess, iron is toxic to cells. Consequently, its level in cells is tightly regulated. Mammalian cells maintain stable cytosolic concentrations of free iron both by regulating their uptake of iron via the transferrin receptor (TfR) and by sequestering intracellular iron into ferritin. Ferritin synthesis is induced by iron through a mechanism which shifts latent ferritin mRNA to actively translating polysomes. TfR synthesis is decreased by iron, which promotes destabilization of TfR mRNA. The coordinate regulation of ferritin and TfR synthesis by iron is controlled by iron responsive elements, or IREs. These IREs are located in the 5'- and 3'-untranslated regions of ferritin and TfR mRNAs, respectively. The IREs form a characteristic stem-loop structure and bind cytosolic proteins, the iron responsive proteins or IRE-BPs. Two such proteins, IRE-BP Bl and IRE-BP B2, have been characterized in part. The proposed experiments are aimed at determining the mechanisms through which ferritin and TfR mRNAs are coordinately regulated by the IRE-BPs. The first specific aim is to isolate an IRE-BP cDNA by screening a rat liver lambda cDNA library with oligonucleotides derived from amino acid sequences of IRE-BP, the principal rat IRE-BP. The cDNA sequence will be determined and the deduced amino acid sequence will be compared to amino acid sequences of other proteins to identify conserved regions of the IRE-BP that may be functionally important in RNA-protein recognition. The second specific aim is to study the expression of IRE-BP B1 in rat hepatoma cells. The IRE-BP cDNA will be used to determine if IRE-BP B1 expression responds to changes in intracellular iron and/or heme levels and to determine if transcriptional or post-transcriptional mechanisms are involved in the regulation of IRE-BP B1 synthesis. The third specific aim is test the predicted secondary of the ferritin and TfR IREs by synthesizing IRE RNAs containing altered nucleotide sequences and testing these mutants for IRE-BP B1 binding. These studies will permit the correlation of IRE structure with biological function. The fourth specific aim is to determine the function of a second protein in rat liver, IRE-BP B2. IRE-BP B2 protein from rat liver will be isolated and sequenced, and its function and the functional determinants in its structure will be characterized. The fifth and last specific aim is to determine if there are other factors involved in the regulation of TfR mRNA by iron. A cell-free system will be developed to identify polysomal or cytosolic factors other than IRE-BPS, that may specifically affect the degradation of TfR mRNA.