Quantitative Analysis Of The Read-through Efficiency Of UGA In Escherichia Coli With Green Fluorescent Protein

Selenium is an essential element for mammals. Many of selenoenzymes areinvolved in redox reactions with selenocysteine acting as an essential component ofcatalytic cycle, such as glutathione peroxidase, thioredoxin reductase, iodothyroninedeiodinase and formate dehydrogenase. Interestingly, selenocysteine is encoded withUGA codon and incorporated into the polypeptide co-translationally. Thus,selenocysteine is named as the twenty-first proteinogenic amino acid. Since UGAcodon remains its routine function in tissues expressing selenoproteins, there is aspecial regulatory mechanism for the incorporation of selenocysteine.In prokaryotes, the incorporation of selenocysteine requires four gene products:on tRNA and three proteins. In addition, recognition of UGA as a selenocysteinscodon depends on secondary mRNA structures, i.e. stem loops called SECIS(Selenocysteine Inserting Sequence), which locates 11 nucleotides downstream ofUGA codon. The SECIS element from prokaryotes is inside the open reading frameof m RNA, while eukaryotic SECIS is in the 5'-or 3'-untranslated region.Furthermore, the bacterial SECIS is species-specific, while the SECIS elements ineubacteria and eukaryotes are somewhat less species-restricted.Glutathione S-transferases (GST) constitute a family of multifunctional proteinsable to detoxify endogenous and xenobiotic electrophiles by conjugation withglutathione (GSH). In the active site of GST's, the catalytic residue is typically eitherTyr or Ser, presumed to act by inducing and stabilizing a reactive thiolate at theconjugating sulfur of the GSH substrate.Both GST and GPX have glutathione-binding sites and thioredoxin-foldstructure, but to a great extent due to the use of different catalytic residues, i.e. Tyr(or Ser) on one hand and Sec on the other, they have completely different catalyticmechanisms. Thus, we tried to achieve GST-Sec, using the selenoprotein synthesismachinery of E. coli. In addition, we constructed a green fluorescent protein forquantitaviely detecting the read-through efficiency of UGA codon, and examined theinfluence of co-expressing of selA, selB and selC genes on the read-throughefficiency.GFP as a reporter gene quantitatively detecting the read-through efficiency ofselenocysteineSince GFP was reported as reporter gene in 1994, it has been used extensivelythroughout the biological science, such as fusion tag, reporter, protein interaction andfluorescent energy resonance transfer. GFP is able to autocatalytically form afluorescence without external agents other than oxygen.The Tr-GFP fusion was constructed with inserting sel-gfp gene fragment intothe multiple clone sites downstream of thioredoxin (Tr) in plasmid pET-32b. Therewas different combination of UGA codon and SECIS element in the DNA linkerbetween thioredoxin and GFP. When UGA codon was read through, GFPdownstream would be translated. Then the fluorescence intensity of GFP can be usedfor detecting quantitatively the read-through efficiency of UGA codon. The resultsshowed that the read-through efficiency of UGA codon was increased to 1.32-fold inthe presence of SECIS element compared to that without SECIS element, and theefficiency was futher increased up to 2.0-fold when GFP fusion was co-expressedwith selA, selB and selC genes. These results suggest that GFP can be served as arapid and convenient tool compared to β-galactosidase for measuring theread-through efficiency of UGA codon in intact E. coli cells, and the potentialapplication of GFP in measuring the Opal suppression in mammalian cell culture andtissue samples.