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。

自然中痕量元素硒通过“丝氨酸途径”间接掺入蛋白质多肽链成为硒蛋白：硒代半胱氨酸(Sec)由终止密码子UGA编码，tRNA(Sec)经SerRS催化与Ser连接；Ser-tRNA(Sec)经由SelA催化，将丝氨酸中S替换为Se，成为Sec-tRNA(Sec)被招募至70S核糖体，受SelB和SECIS元件识别和协助，掺入延伸的多肽链中。目前，掺硒效率低、硒蛋白产量低，制约硒蛋白研究和硒生物学发展。本研究主要研究“丝氨酸途径”涉及的相互识别与结合作用，解析硒蛋白原核生物合成的过程和调控并提供新思路。以大鼠细胞质型硫氧还蛋白还原酶(TrxR1)为原核掺硒模式蛋白，基于分子互作对接的结构信息，通过随机突变、定点突变和抗性筛选，在体外验证tRNA(Sec)关键位点和新型SECIS元件。将随机突变及定点突变的tRNA(Sec)所在的pSUABC’转化至BL21(DE3) gor-获得阳性重组菌株，其携带pET-TRSter共表达大鼠硒蛋白TrxR1；使用2’,5’ ADP-Sepharose亲和层析和凝胶过滤分离纯化TrxR1；最后利用经典硒依赖型DTNB还原反应测定TrxR1活性，与位点进行关联分析，评价掺硒效率。本研究在大肠杆菌体系中观察到Sec特异性tRNA和mRNA 3’-UTR的SECIS影响并调控Sec高效合成与活性掺入硒蛋白。天然SECIS元件条件下，与野生型相比，E. coli tRNA(Sec)骨架上发生突变，使得新合成的TrxR1活力呈现不同程度的降低：G18、G19 这两个位点突变，使所有TrxR1酶活远低于野生型(<10%)；然而U20A/A21G和G64U/U65C双突变，所对应的TrxR1活力相对较高。一系列突变研究表明，tRNA(Sec)骨架上G18和G19位点对于维持tRNA 稳定性和灵活性发挥了关键作用，位点突变引起tRNA 结构变化会影响tRNA(Sec)与掺硒元件的互作，因此，改造tRNA核苷酸位点可以提高硒蛋白的掺硒效率。后续工作可以聚焦全新tRNA(Sec)骨架和全新SECIS元件，通过与原核掺硒机器完美匹配，实现更高掺硒效率。本研究寻找新型tRNA(Sec)骨架和SECIS元件，揭示大肠杆菌掺硒机器的掺硒和调控，为获得全长硒蛋白的合成技术提供参考。

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