SYNTHESIS OF SELENIUM CONTAINING PROTEINS

含硒蛋白质的合成

• 批准号: 6636442
• 负责人: RAYMOND F GESTELAND
• 金额: $ 31.34万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636442
• 关键词: Caenorhabditis elegans; alternatives to animals in research; aminoacid analog; cysteine; genetic mapping; genetic translation; messenger RNA; nucleic acid sequence; protein biosynthesis; selenated sulfur aminoacid; selenoprotein; transfer RNA; transposon /insertion element; yeast two hybrid system

Selenium plays a key metabolic role as selenocysteine the 21st amino acid that is encoded by the genetic code. Of the 30-50 selenocysteine containing proteins in mammalian cells, only 10 or so are well characterized. In most cases the amino acid is at the active site and plays a key role in the catalytic reaction and often plays a key role in protection from oxidative insult. Selenocysteine is encoded by UGA, which in most mRNAs is the signal for stop. A complex stem loop in the 3' untranslated region of these special mRNAs (SECIS element) somehow reprograms the ribosome to read UGA as selenocysteine via a tRNA specific for this amino acid. The mechanism of insertion in eukaryotes is not understood though it is clearly different than in prokaryotes. We will use the power of C. elegans genetic system to find the genes involved in the insertion pathway to work out the mammalian homologs. Both up and down the mutants will be sought using a vector with a selectable gene and a GFP screening gene each engineered to have a UGA codon that needs to be read by the selenocysteine insertion mechanism. Mutants will be mapped, their genes identified and products characterized. The structure of the SECIS element and its interactions with any proteins in the pathway will be studied. Six specific aims are to: (1) establish a C. elegans selection system for up and down mutants of Sec synthesis; (2) map and characterize identified mutant genes; (3) identify interacting components; (4) determine SECIS structure, including using NMR; (5) test RNA binding of putative selB homolog; (6) test models for eukaryotic Se insertion.

硒作为第21代硒代半胱氨酸发挥着关键的代谢作用 由遗传密码编码的氨基酸。 30-50个硒代半胱氨酸 哺乳动物细胞中含有蛋白质，但只有 10 种左右得到了很好的表征。 在大多数情况下，氨基酸位于活性位点并在 催化反应，通常在防止氧化方面发挥关键作用 侮辱。硒代半胱氨酸由 UGA 编码，在大多数 mRNA 中，UGA 是以下信号： 停止。这些特殊 mRNA 的 3' 非翻译区中存在复杂的茎环 （SECIS 元素）以某种方式重新编程核糖体，将 UGA 读取为硒代半胱氨酸 通过对该氨基酸具有特异性的 tRNA。插入机制 真核生物尚未被理解，尽管它与真核生物明显不同 原核生物。我们将利用线虫遗传系统的力量来寻找 参与插入途径的基因以计算出哺乳动物同源物。 将使用具有可选择的载体的载体来寻找向上和向下的突变体 基因和 GFP 筛选基因各自被设计为具有需要的 UGA 密码子 由硒代半胱氨酸插入机制读取。突变体将被映射， 他们的基因被识别，产品被表征。 SECIS 的结构 将研究元素及其与途径中任何蛋白质的相互作用。 六个具体目标是：（1）建立线虫选择体系 以及Sec合成的下调突变体； (2) 绘制并表征已识别的突变体 基因； (3) 识别相互作用的组件； (4)确定SECIS结构， 包括使用核磁共振； (5)测试推定的selB同源物的RNA结合； (6)测试 真核 Se 插入模型。