UGA-mediated selenium incorporation into glutathione peroxidase 1 and green fluorescent protein

A UGA codon and a selenocysteine (Sec, or U) insertion sequences are the only established mRNA elements necessary for Sec incorporation. Using lacZ fusion genes, we found that the 3'UTR of neither glutathione peroxidase 1 (gpx1) nor gpx4 was able to direct Sec insertion at the engineered UGA codon in lacZ. In contrast, we observed efficient expression of recombinant GPX1. These data suggest that besides a UGA codon and a SECIS element, other components of the mRNA may be critical for efficient Sec incorporation.; For FLAG-tagged GPX1 (F-GPX1) and three GPX1/GFP fusion constructs, we found that the spacing between the UGA and AUG-start affects the efficiency of Sec insertion at N-terminal positions of GPX1; and the spacing between the UGA and SECIS affects the efficiency of Sec insertion at the C-terminal positions of GPX1. Efficient Sec insertion appears to require that the UGA to be {dollar}>{dollar}21 nt from the AUG-start and {dollar}>{dollar}204 nt from the SECIS. Interestingly, increasing the spacing between the UGA and SECIS reduced Sec insertion at UGA codons in the middle of the coding region, suggesting that there might be an optimum spacing between the UGA and SECIS. Results with Sec insertion at S195U in these constructs strongly suggest that S195U may have a unique context that enhances Sec incorporation.; We developed a green fluorescent protein (GFP) with Sec at 147, the first unnatural selenoprotein that accounts for about 20% of the host cell's total selenoproteins. Sec insertion at 10 other engineered UGA codons in gfp, however, was either very low or undetectable. In contrast, every one of the 11 F-GPX1s studied was expressed. On average the F-GPX11 accounted for more than 20% of cell's total selenoproteins. This, for the first time, reveals the dramatic difference in Sec insertion into a natural, and an unnatural selenoprotein.; Even when {dollar}sp7sp5{dollar}Se incorporation into some of the Sec-containing GFPs was only observed after overexposure, the GFP fluorescence of cells expressing these proteins was readily detectable. The best expressed one, with Sec at C147U, could be used as a convenient and sensitive tool in selenium biology research. Based on quantitation by immunoblotting, replacement of Thr65 by Sec may have improved the specific fluorescence of GFP considerably.; In summary, in this project we demonstrated that the UGA position affects Sec insertion. The UGA context may be critical for efficient Sec insertion. Sec is much more efficiently inserted into a natural selenoprotein than into an unnatural selenoprotein. The novel Sec-containing GFPs could be used as reporters in selenium biology research.