Molecular Mechanisms of Selenocysteine incorporation in Bacterial Bakterial Translation

当硒代半胱氨酸结合序列（SECIS）存在于mRNA中时，硒代半胱氨酸在被Sec-tRNASec解码的UGA密码子（通常导致终止）中被结合到核糖体上的蛋白质中。该项目旨在了解硒代半胱氨酸在大肠杆菌系统中掺入的分子机制。将动力学研究含有secis的mRNA和与核糖体的三元复合物SelB·GTP·Sec-tRNASec的组装，监测复合物组分不同位置的荧光染料。我们将研究GTPase在三元复合物中激活SelB的机制，以及mRNA识别元件UGA密码子和SECIS对SelB的调控作用。SelB和SECIS荧光组之间的荧光共振能量转移（FRET）将研究SelB从核糖体·SECIS- mrna复合物解离的时间和机制。对体内硒化半胱氨酸掺入效率低的原因进行研究。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。与MPI Göttingen的H. Stark和M. Wahl合作，将通过冷冻电子显微镜和晶体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.