Molecular Mechanisms of Selenocysteine incorporation in Bacterial Bakterial Translation

细菌翻译中硒代半胱氨酸掺入的分子机制

• 批准号: 5425290
• 负责人: Professorin Dr. Marina V. Rodnina
• 金额: --
• 依托单位: Max-Planck-Institut für Multidisziplinäre Naturwissenschaften
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2004
• 资助国家: 德国
• 起止时间: 2003-12-31 至 2006-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5425290?language=en
• 关键词: Molecular; Mechanisms; Selenocysteine; incorporation; Bacterial

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.

当通常导致终止的UGA密码子被Sec-tRNASec解码时，硒代半胱氨酸被掺入核糖体上的蛋白质中，前提是mRNA中存在硒代半胱氨酸掺入序列（SECIS）。本项目旨在了解硒代半胱氨酸在大肠杆菌中的分子机制。coli系统。SECIS-含有mRNA和三元复合物SelB·GTP·Sec-tRNASec与核糖体的组装将进行动力学研究，监测复合物组分不同位置的荧光染料。将研究GTdR在三元复合物中激活SelB的机制及其通过mRNA识别元件UGA密码子和SECIS的调节。SelB从核糖体·SECIS-mRNA复合物中解离的时间和机制将通过SelB和SECIS中的荧光基团之间的荧光共振能量转移（FRET）来研究。将研究体内硒代半胱氨酸掺入效率低的原因。实验将使我们能够识别反应中间体，并确定相应的过渡动力学。与H合作。Stark和M. Wahl，MPI Göttingen，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。