Genetic and biochemical analysis of selenoprotein biosynthesis in Archaea (B 11)

含硒代半胱氨酸的蛋白质是第21个协同插入的氨基酸，存在于生命的所有三个领域。硒代半胱氨酸的通用密码子是mRNA上的蛋白石（终止）密码子UGA。为了介导终止密码子后退到感觉密码子中，需要硒蛋白mRNA上存在二级结构（SECIS元件）。硒代半胱氨酸的生物合成机制及其掺入维生素多肽的机制在细菌如E。大肠杆菌，令人惊讶的是，在古细菌和真核系统的理解存在很大的差距。从已知的情况来看，后两个结构域的硒蛋白生物合成过程存在着惊人的相似性。因此，在一个系统中获得的见解可能适用于另一个系统。海沼甲烷球菌是一种嗜温、生长迅速的产甲烷古菌，是研究海沼甲烷菌硒蛋白生物合成机制的理想模式菌。它是唯一已知的含硒蛋白的古菌，并建立了一个简单的遗传分析系统。此外，在一定条件下，其硒蛋白均为非必需的。在本研究过程中，对硒代半胱氨酸的生物合成途径和M.将通过遗传和生物化学方法对海沼生进行研究。对不能合成硒蛋白的突变体进行表型分析，以及对相关基因产物进行生化分析，除了这些新发现的明显价值外，还将使我们更好地理解不易处理的真核系统。

Proteins that contain selenocysteine, the 21st co-translationally inserted amino acid, arepresent in members of all three domains of life. The universal codon for selenocysteine is theopal (stop-) codon UGA on the mRNA. To mediate receding of the stop codon into a sensecodon, the presence of a secondary structure on the selenoprotein mRNA (the SECIS element)is required. While the mechanism of selenocysteine biosynthesis and its incorporation intonascent polypeptides is well understood in bacteria such as E. coli, surprisingly large gapsexist in the understanding of the archaeal and the eukaryal system. From what is known it isapparent that striking similarities exist in the process of selenoprotein biosynthesis of thelatter two domains. Thus, insights gained in one system are potentially applicable in the othersystem. Methanococcus maripaludis, a mesophilic, fast growing methanogenic archaeon is anexcellent model to study the mechanisms of selenoprotein biosynthesis in Archaea. It is theonly known selenoprotein-containing archaeon for which a facile system for genetic analysesis established. Furthermore, all of its selenoproteins were shown to be non-essential undercertain conditions. In the course of the proposed research the pathway of selenocysteinebiosynthesis and incorporation of M. maripaludis will be investigated by genetic andbiochemical approaches. Phenotypical analyses of mutants unable to synthesizeselenoproteins as well as biochemical analyses of gene products involved, will, beside theobvious value of the new insights, also lead to a better understanding of the less tractableeukaryal system.