大肠杆菌tRNASec关键核苷酸位点

在原核生物中,硒蛋白合成需要tRNA~(Sec) (SelC)与硒代半胱氨酸合成(Sec synthase, SelA)、硒代半胱氨酸特异性延伸因子(Sec-specificelongationfactor,SelB)之间相互作用。【目的】基于大肠杆菌掺硒机器,寻找tRNA~(Sec)骨架上关键核苷酸位点,为解决硒蛋白目前面临的掺硒效率较低、产量低的问题提供新思路。【方法】以大鼠细胞质型硫氧还蛋白还原酶(thioredoxinreductase1,TrxR1)为掺硒模式蛋白为定点突变tRNA~(Sec),转化至BL21 (DE3) gor-获得阳性重组菌株(携带pET-TRSter/pSUABC’),用于表达大鼠硒蛋白TrxR1,然后使用2￠,5￠ADP-Sepharose亲和层析和凝胶过滤两步法分离纯化TrxR1,最后利用经典硒依赖型DTNB还原反应测定TrxR1的酶活,分析关键核苷酸位点,评价掺硒效率。【结果】在存在SECIS元件的前提下,当SelA、SelB、tRNA~(Sec)共表达时,与野生型相比,携带突变型tRNA~(Sec)所共表达的TrxR1酶活力呈现不同程度的降低,其中E.colitRNA~(Sec)的G18、G19这两个位点的所有的TrxR1酶活远低于野生型(10%);然而,a26和b7的酶活相对较高。【结论】E. coli 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,因此有望通过改造tRNA核苷酸位点来提高硒蛋白的掺硒效率。

Key nucleotide sites of Escherichia coli tRNASec

In prokaryotes, selenoprotein synthesis requires the interaction between selenocysteine (Sec)-specific tRNA^Sec (SelC) and selenocysteinyl tRNA synthase (SelA) or Sec-specific elongation factor (SelB).Objective] Based on the Sec insertion machinery in Escherichia coli, we aimed at finding key sites of tRNA^Sec scaffold and providing a new way to solve the problems of low Sec insertion efficiency and low yield of selenoprotein.Methods] We used rat cytoplasmic thioredoxin reductase (thioredoxin reductase 1, TrxR1) as a model selenoprotein. First, we constructed tRNA^Sec by site-directed mutagenesis, and transformed them to BL21 (DE3) gor-to obtain a positive recombinant strain (carrying pET-TRSter''/pSUABC), which was used to express rat cytoplasmic thioredoxin reductase 1 (TrxR1). Then TrxR1 was purified using 2'',5''-ADP Sepharose affinity chromatography and gel filtration. The enzyme activities of TrxR1 were determined by the classical Se-dependent DTNB reduction assay to analyze the key nucleotide sites and evaluate the Sec incorporation efficiency.Results] When tRNA^Sec co-expressed with SelA and SelB in the presence of the bacterial SECIS element, the activities of TrxR1 decreased at varying degrees compared with wild type. Among them, the enzyme activities of all G18 and G19 mutants were much lower than that of wild type (<10%). However, the enzymatic activities of a26 and b7 were relatively high.Conclusion] G18 and G19 nucleotide sites of E. coli tRNA^Sec may play a vital role in maintaining the stability and flexibility of tRNA^Sec. Site-directed mutations of tRNA^Sec causing the conformational change may affect the interaction between tRNA^Sec and Sec elements. Thus, it is possible to improve the Sec insertion efficiency via proper modification on tRNA nucleotide sites.