Mechanism of selenoprotein synthesis in bacteria

在细菌中，硒代半胱氨酸的UGA终止密码子的后退需要一个专门的翻译因子SelB，一个非经典的Sec-tRNASec和mRNA中的硒代半胱氨酸掺入序列（SECIS）。本项目旨在了解硒代半胱氨酸插入蛋白质的分子机制。将测量体内UGA重新编码的速率，并开发体外系统，其允许硒代半胱氨酸以体内速度和特异性掺入。三元复合物SelB-GTP-Sec-tRNASec与核糖体的相互作用的动力学机制将使用生物化学和生物物理技术的组合进行研究。SelB的GTP水解的硒代半胱氨酸插入的作用将得到解决。将研究Sec-tRNASec的积极校对，这可能是体内硒代半胱氨酸掺入效率低的原因之一。与H合作。Stark和M. Wahl，MPI Göttingen，功能性核糖体-SelB复合物和SelA-Sec-tRNASec复合物的结构将分别通过冷冻电子显微镜和晶体学进行研究。

In bacteria, the receding of a UGA stop codon for selenocysteine requires a specialized translation factor SelB, a non-canonical Sec-tRNASec, and a selenocysteine incorporation sequence (SECIS) in the mRNA. The present project aims at understanding the molecular mechanisms of selenocysteine insertion into proteins. The rate of UGA recoding in vivo will be measured and an in vitro system developed which allows selenocysteine incorporation at in vivo speed and specificity. Kinetic mechanisms of the interaction of the ternary complex SelB-GTP-Sec-tRNASec with the ribosome will be studied using a combination of biochemical and biophysical techniques. The role of GTP hydrolysis by SelB for selenocysteine insertion will be addressed. Aggressive proofreading of Sec-tRNASec, which is probably one reason for the low efficiency of in vivo selenocysteine incorporation, will be investigated. In collaboration with H. Stark and M. Wahl, MPI Göttingen, the structure of functional ribosome-SelB complexes and of the SelA-Sec-tRNASec complex will be studied by cryoelectron microscopy and crystallography, respectively.