Directed Evolution Of Escherichia Coli TRNA^Sec In Vitro

Selenocysteine as the 21st natural amino acid,encoded by stop codon UGA,is incorporated into the polypeptide chain through a complex mechanism to form selenoprotein.Selenoproteins widely exist in organisms and play a role in regulating redox stress,influencing apoptosis and maintaining cell growth and proliferation.In prokaryotes,selenocysteine synthesis requires the interaction of tRNASec,Sec synthase(SelA)and Sec-elongation factor(SelB).Firstly,SerRS catalyzes tRNASec to produce Ser-tRNASec;Secondly,SelA catalyzes Ser-tRNASec to form Sec-tRNASec.Then,SelB recognizes and combines Sec-tRNASec and SECIS element,and finally completes the Sec incorporation.At present,the main problems in selenoprotein research are the low efficiency of Sec incorporation and the low yield of selenoprotein.The mechanism of Sec incorporation in the prokaryotic system remains to be solved.In this study,based on the Sec incorporation of E.coli in a prokaryotic system,we started with the nucleotide sites of the interaction between E.coli tRNASec and SelA and SelB and searched for the key nucleotide sites on the tRNASec scaffold.Specifically,the following work has been carried out:1)Firstly,we constructed tRNASec mutants using direct-site mutation.2)Then the mammalian cytoplasmic thioredoxin reductase 1(TrxR1)was expressed by BL21(DE3)gor-(pET-TRSter/pSUABC)strain,and the selenoprotein TrxR1 was purified by 2'5'ADP-Sepharose affinity chromatography.3)Nest,we used DTNB assay to test the enzyme activity of TrxR1 to evaluate the Sec incorporation efficiency and thus completing directed evolution.4)Finally,we expressed and purified SelA protein,and used the EMSA method to test the bonding strength of SelA and tRNASec mutants.The results showed that:When co-expressing SECIS elements with SelA,SelB,and tRNASec,the activity of most TrxR1 mutants decreased at different degree comparing to the wild type.Among them,the activity of the mutants TrxR1 of the site G18 and G19 in E.coli tRNASec is far less than wild type(<10%).C15,C16,U20,A21 are the bonding sites with SelA,while C50,A51,C56,U63,G64,and U65 may be the binding site of SelB.Among them,the specific activity of the U20C mutant was 10%higher than that of wild type.When TrxR1 was expressed in the absence of selA,selB,and selC,the Sec incorporation efficiency of most mutants decreased,while a few mutants increased.Conclusion:Some key nucleotides in E.coli tRNASec scaffold for maintaining its stability and flexibility which affects the Sec insertion machinery.G18 and G19 may be the key nucleotide sites to maintain the tertiary structure of E.coli tRNASec.C15,C16,U20,and A21 are binding sites to SelA.C50,A51,C56,C59,U63,G64 and U65 may be binding sites to SelB.Different tRNASec mutants can bind different quantities of SelA and SelB,which may be due to the influence of structural changes of mutants on the binding degree of SelA and SelB.Significance:The key nucleotide sites of E.coli tRNASec regulating Sec insertion mechanism were proposed for the first time in this study.The recognition and regulation mechanism between prokaryotic Sec insertion elements tRNASec and trans-acting factors of SelA and SelB were preliminarily revealed,which provided new insights for bacterial engineering to modify tRNASec to prepare full-length selenoproteins.