Molecular Mechanisms of Selenocysteine incorporation in Bacterial Bakterial Translation

当通常导致终止的UGA密码子被SEC-tRNASec解码时，如果mRNA中存在硒半胱氨酸掺入序列(SECIS)，硒半胱氨酸就会被掺入核糖体上的蛋白质中。该项目旨在了解硒半胱氨酸在大肠杆菌系统中掺入的分子机制。含SECIS的mRNA和三元络合物SELB·GTP·SEC-tRNASec与核糖体的组装将被动力学研究，监测复杂组分不同位置的荧光染料。我们将研究三元复合体中SELB的GTPase激活机制，以及它受mRNA识别元件UGA密码子和SECIS的调节。用荧光共振能量转移技术(FRET)研究SELB和SECIS中荧光基团之间的解离时间和解离机理。体内硒半胱氨酸掺入效率低的原因将被调查。这些实验将使我们能够识别反应中间体，并确定各自转变的动力学。在H.Stark和M.Wahl，MPI Göttingen的合作下，将用低温电子显微镜和结晶学研究功能性核糖体-SELB复合物和SELA·SEC-tRNASec复合物的结构。

Selenocysteine is incorporated into protein on the ribosome when UGA codons, which usually lead to termination, are decoded by Sec-tRNASec, provided a selenocysteine incorporation sequence (SECIS) is present in the mRNA. The project aims at understanding molecular mechanisms of selenocysteine incorporation in the E. coli system. The assembly of SECIS-containing mRNA and the ternary complex SelB·GTP·Sec-tRNASec with the ribosome will be studied kinetically, monitoring fluorescent dyes at various positions of the complex components. The mechanism of GTPase activation of SelB in the ternary complex and its regulation by the mRNA recognition elements, UGA codon and SECIS, will be investigated. Timing and mechanism of SelB dissociation from the ribosome·SECIS-mRNA complex will be studied by fluorescence resonance energy transfer (FRET) between fluorescent groups in SelB and SECIS. Reasons for the low efficiency of in vivo selenocysteine incorporation will be investigated. The experiments will allow us to identify reaction intermediates and determine the kinetics of the respective transitions. 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.