基于大肠杆菌掺硒机器的新型tRNA(Sec)/SECIS元件的发现与调控机制

Selenocysteine (Sec) is encoded by the UGA stop codon and is incorporated into the growing polypeptides to generate selenoproteins through an intrinsic mechanism. So far, the molecular interactions and recognitions among Sec-insertion factors/elements are complicated but crucial for site-specific Sec insertion and selenoprotein production. Currently, however, the regulation process of selenoprotein biosynthesis remains unclear, and the efficient preparation of full-length selenoprotein is still challenging. In the present study, the significant effects of tRNA(Sec) and SECIS element on high-efficiency biosynthesis and Sec incorporation were observed. Herein, with the E. coli selenocysteine insertion machinery, the main objects of this proposal are: 1) To search and find key nucleotide residues of tRNA(Sec) scaffold in relation to Sec incorporation, based on the molecular interaction and docking information, by means of random mutagenesis and site-directed mutagenesis. 2) To search for novel SECIS element in compatible with the E. coli selenocysteine insertion machinery. 3) To validate the novel tRNA(Sec) and SECIS element by using the in vitro screening system and rat TrxR1 as model selenoprotein. 4) To inquire a novel methodology for high-efficiency Sec-incorporation and full-length selenoprotein preparation, and to elucidate the function and regulation mechanism of E. coli selenocysteine insertion machinery.

硒代半胱氨酸(Sec)由终止密码子UGA编码，通过复杂机制掺入多肽链、生成硒蛋白，涉及到一系列辅助因子和掺硒元件的相互识别与结合。目前，尚不能洞悉硒蛋白生物合成的调控过程，还无法高效制备全长硒蛋白。申请人在大肠杆菌体系中观察到Sec特异性tRNA和mRNA 3‘UTR的SECIS可能影响和调控Sec的高效合成与活性掺入。本研究拟 1) 在大肠杆菌系统中，基于分子互作、分子对接所获得的结构信息，通过随机突变和定点突变等手段，发现大肠杆菌tRNA(Sec)骨架上影响掺硒的关键核苷酸位点；2) 寻找与大肠杆菌掺硒机器相匹配的新型SECIS元件；3) 应用抗性筛选系统和大鼠硒蛋白TrxR1为模型，在体外验证tRNA(Sec)关键核苷酸位点和新型SECIS元件；4) 获得高效生物合成全长硒蛋白的技术方法，揭示大肠杆菌掺硒机器的掺硒作用和调控机制。