SYNTHESIS OF SELENIUM CONTAINING PROTEINS

含硒蛋白质的合成

• 批准号: 6090313
• 负责人: RAYMOND F GESTELAND
• 金额: $ 32.89万
• 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/6090313
• 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.

硒作为硒仿制半胱氨酸的关键代谢角色21 由遗传密码编码的氨基酸。在30-50硒代半胱氨酸 在哺乳动物细胞中含有蛋白质，只有10个左右的表征。 在大多数情况下，氨基酸在活性部位，在 催化反应，通常在保护免受氧化方面起关键作用 侮辱。硒代半胱氨酸由UGA编码，在大多数mRNA中，这是 停止。这些特殊mRNA的3'未翻译区域中的复杂茎回路 （SECIS元素）以某种方式重新编程核糖体以将UGA读为硒代半胱氨酸 通过针对该氨基酸的特异性tRNA。插入机制 真核生物尚不清楚，尽管它与 原核生物。我们将利用秀丽隐杆线虫遗传系统的力量找到 参与插入途径的基因来制定哺乳动物同源物。 使用可选的向量，都将在突变体上下寻求 基因和一个GFP筛选基因每个设计，都有一个需要的UGA密码子 可以通过硒仿半半胱氨酸插入机制阅读。突变体将被映射， 它们的基因确定和产品的特征。 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）建立秀丽隐杆线虫选择系统 和SEC合成的下降突变体； （2）映射和表征已识别的突变体 基因； （3）确定相互作用的组件； （4）确定secis结构， 包括使用NMR； （5）假定的SELB同源物测试RNA结合； （6）测试 真核SE插入的模型。