(A) Schematic representation of eGPx1 mRNA. The positions of the start (AUG), Sec (UGA) and stop (UAA) codons, and the SECIS element in the 3′-UTR are shown. (B) eGpx1 SECIS element. Core highlights the mutations made in the SECIS core. (C) Expression of GFP-eGPx1 fusion in HEK 293 cells. Cells were transfected with the pEGFP-C3 vector, pEGFP-eGPx1 construct or construct with mutations in the SECIS core (pEGFP-eGPx1core, see B), labeled with ⁷⁵Se and analyzed as given in SOM. Selenoprotein patterns were visualized with a PhosphorImager on SDS-PAGE gels. (D) Western blot analysis of samples shown in C with anti-GFP antibodies. Arrows show the positions of GFP and GFP-eGPx1. (E) Expression of GFP-ep22 fusion protein in HEK 293 cells. Cells were transfected with the pEGFP-C3 vector or pEGFP-ep22 construct, labeled with ⁷⁵Se and analyzed as in C. (F) Western blot analysis of samples shown in E with anti-GFP antibodies. Arrows show the positions of GFP and GFP-ep22. (G) Expression of GFP-eSelW2 fusion protein in HEK 293 cells. Arrow shows the position of the GFP-eSelW2 fusion selenoprotein. Molecular masses of protein standards (in kDa) are shown on the left.

(A) Expression of GFP-eTR1 in HEK 293 cells. Cells were transfected with pEGFP-C3 vector, a GFP-eTR1 construct containing a single UGA codon at the natural Sec position 497 (pEGFP-497) or constructs that had UGA at unnatural positions 246 (pEGFP-246), 441 (pEGFP-441) or 494 (pEGFP-494). Cells were analyzed as described in Figure 1C. (B) Western blot analysis of samples shown in A with anti-GFP antibodies. (C) Cells were transfected with pEGFP-C3 vector, a GFP-eTR1 construct containing a single UGA codon at the natural Sec position 497 (pEGFP-497) or constructs that had UGA at unnatural positions 467 (pEGFP-467), 478 (pEGFP-478) or 489 (pEGFP-489). (D) Western blot analysis of samples shown in C with anti-GFP antibodies. (E) Summary of experimental evidence for Sec and Cys insertion in eTR1. The positions of Cys insertion (corresponding to termination in mammalian cells) are shown by blue, and Sec insertion by red lines. Position 478 supported low level Sec insertion. (F) Cells were transfected with pEGFP-C3 vector, a GFP-eTR1 construct containing a single UGA codon at the natural Sec position 497 (pEGFP-497) or constructs containing a 3'-UTR segment of Toxoplasma SelT and UGA at position 420 (pEGFP-420toxo) or 497 (pEGFP-497toxo). (G) Cells were transfected with pEGFP-C3 vector or with ep22 constructs in which UGA corresponded to positions 190 (pEGFP-ep22) or 44 (pEGFP-ep22-44). Arrows show the positions of GFP and full-size GFP-eTR1 or GFP-ep22.

(A) Structures of E. crassus tRNAs. Sec tRNA, Cys tRNAs with UCA and GCA anticodons and a mitochondrial Trp tRNA are shown. Anticodons are highlighted in red (UCA) or blue (GCA). (B) Expression of GFP-eTR1 in HEK 293 cells. Cells were transfected with pEGFP-C3 vector (lane 1), pEGFP-eTR1 (lane 2) or constructs with multiple UGA to UGC mutations in which the number indicates the amino acid residue for which the UGA codon is retained: pEGFP-68 (lane 3), pEGFP-420 (lane 4) and pEGFP-497 (lane 5). Cells were analyzed as described in Figure 1C. Arrow shows the position of the GFP-eTR1 fusion selenoprotein. (C) Western blot analysis of samples shown in B with anti-GFP antibodies. Arrows show the positions of GFP and truncated and full-size GFP-eTR1. Asterisks show the position of truncated GFP-eTR1 fusions in lanes 4 and 5. (D) Partially purified eTR sample analyzed by 2D-PAGE and stained with Coomassie Blue. (E) Visualization of the ⁷⁵Se-labeled sample shown in D with a PhosphorImager. The spots of eGR are indicated by a blue oval (in D) and of eTR by red ovals (in D and E).