Quantitative Analysis The Read-through Efficiency Of Sec In E.coli With β-galactosidase Reporter Gene

Selenium is an essential element for mammals. Many selenoenzymes areinvolved in redox reactions with selenocysteine acting as an essential componentof the catalytic cycle, such as glutathione peroxidase, thioredoxin reductase,iodothyronine deiodinase and formate dehydrogenase. Low selenium status hasbeen associated with numerous diseases, e.g. viral infection, cataract, reproductiondysfunction, mood disorders, and cardiovascular diseases. Interestingly,selenocysteine is encoded by the UGA codon and is incorporated into polypeptidesco-translationally. Thus, selenocysteine is considered the twenty-firstproteinogenic amino acid. Since the UGA codon retains its routine function intissues expressing selenoproteins, there is a special regulatory mechanism for theincorporation of selenocysteine. In prokaryotes, the incorporation ofselenocysteine requires four gene products: one tRNA and three proteins. Inaddition, recognition of UGA as a selenocysteins codon depends on secondarymRNA structure, i.e. stem loops called SECIS (Selenocysteine Inserting Sequence),which is located 11 nucleotides downstream of the UGA codon. The SECISelement from prokaryotes is contained within the open reading frame of mRNA,while eukaryotic SECIS is in the 5'-or 3'-untranslated region. Furthermore, thebacterial SECIS is species-specific, while the SECIS elements in eubacteria andeukaryotes are somewhat less species-restricted.The expression efficiency of Sec is only 1-3% of that of normal aminoacids in Escherichia coli, therefore, the selenoprotein is faintly expressed inorganism. Thioredoxin reductases is the first eukaryotic selenoprotein, which isexpressed in Escherichia coli. Its structure characteristic allows the introduceableSECIS to locate at 3'-UTR, and the Sec of thioredoxin reductases is at the lastsecond position from C terminus, thus there is no effect on the dimensionalstructure of designed protein. The first one, that made the SECIS locate in targetprotein ORF and succeede in the expression, is the mutant of Citrus PHGPX. Ourteam also successfully express the heterogenous selenocysteine-containingglutathione sulfur-transferase using the special regulatory mechanism for theincorporation of selenocysteine. Even if above proteins coexpressed withpSUABC, the quantity of expressed protein is still low, and it is impossible tosatisfy researchers. So we hope that the expression quantity of selenoprotein wouldbe increased by regulating the expression of sel genes, and simultaneouslyexamined the influence of co-expressing selA, selB and selC genes onread-through efficiency of UGA codon. We utilized the mature and stable β-galactosidase reporter gene system to assess the effect.To assess the influence of selA ,selB and selC genes on the read-throughefficiency of UGA codon, we constructed different expression vectors with thefollowing genes: pBAD-selA, pBAD-selAB, pBAD-selAB(SD), pBAD-selB,pBAD-selB13, pBAD-selB44, pBAD-selC and pEXT-selC, using plasmids withcompatible replicon genes and antibiotics resistance genes.The SelC factor is a selenocysteine-specific tRNA originally charged with aseryl moiety (seryl-tRNASec), which is converted to selenocysteinyl-tRNASec bythe action of selenocysteine synthetase (SelA). SelB is the selenocysteine-specificelongation factor, which achieves specificity by interaction with bothselenocysteinyl-tRNASec and the SECIS element of the mRNA. Thereby thequaternary complex SelB?GTP?Sec-tRNASec?SECIS is formed and Sec may thenbe inserted by SelB on the ribosome into the growing selenopolypeptide at the sitecorresponding to the selenocysteine-encoding UGA codon. When co-expressingwith selC, the read-through efficiency of Sec was increased by 40% compared tothe individual Sec gene. The result is reasonable. However, when co-expressingwith the selA or selB gene, the read-through efficiency of UGA codon wasdecreased by 39% and 89%, respectively. These results suggested that selA andselB did not improve but suppress the read-through of Sec. When co-expressingwith the selB and selC gene, the read-through efficiency was increased by 11%. Atthat level, we added the selA gene, then the read-through efficiency was decreasedby 19%. The co-expressing with the selAB and selC gene made the read-throughefficiency of Sec increase by 19% if the arabinose concentration was decreased. Sowe guess that the SelA has negative function in the process and the SelB is thecrucial regular protein, which would suppress the expression level of SelA in vivo.At the same time, the read-through efficiency of Sec would be sharply descend dueto the strong translation termination resulted from the overexpression of the SelBprotein. In addition, SelB may suppress the initiation of translation. To our joy, theread-through efficiency of Sec was enhanced by 5.46 times when Sec gene wasco-expressed with the selAB and selC gene. However, the read-through efficiencyof Sec was only increased by 2.22 times when Sec was co-expressed withpSUABC under the common conditions accepted by expressing selenoprotein.Obviously our method is better than pSUABC at the read-through efficiency ofSec or selenoprotein expression.