Mechanism of selenoprotein synthesis in bacteria

点击翻译按钮获取中文摘要

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.