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. 2007 Mar 1;21(5):519–524. doi: 10.1101/gad.1490207

Figure 3.

Figure 3.

The insertion of a poly(A) tract upstream of a termination codon reduced the expression. (A) Translation repression and protein destabilization by the insertion of a poly(A) tract upstream of a termination codon. (Left panel) The levels of proteins in W303 cells harboring indicated plasmid were analyzed as described in Figure 1B. (Lane 1) pGPDp-GFP-HIS. (Lane 2) pGPDp-GFP-HIS3-K12PGDGS. (Lane 3) pGPDp-GFP-HIS3-K12. (Lane 4) pGPDp-GFP-2A-HIS3. (Lane 5) pGPDp-GFP-2A-HIS3-K12PG DGS. (Lane 6) pGPDp-GFP-2A-HIS3-K12. (Middle panel) Protein samples prepared from cells harboring pGPDp-GFP-2A-HIS3 (WT) or pGPDp-GFP-2A-HIS3-K12 (K12) were diluted fourfold and analyzed by Western blotting with anti-GFP antibody. (Right panel) The relative levels of each product normalized to the level of GFP-2A-His3 as 100% are shown as the mean values of three independent experiments with standard deviations. (B) The insertion of the poly(A) tract does not affect the mRNA level. W303 cells described in A were cultured, and the levels of reporter mRNAs were determined by Northern blotting with GFP probe. (C) Effect of the number of inserted lysine residues at C terminus on the expression. W303 cells were transformed with a series of reporter genes (pGPDp-Flag-HIS3-Kn) encoding proteins bearing an indicated number of lysine residues at C terminus. The levels of protein products were determined with Western blotting, and numbers immediately below the figure represent the level of protein normalized to the level of His3p as 100%. (D) An amino acid sequence is a determinant to reduce the level of protein produced from mRNA containing the poly(A) tract in a coding region. (Left) The levels of proteins in W303 cells harboring pGPDp-GFP-2A-HIS3 (WT), pGPDp-GFP-2A-HIS3-K12 [K12(AAA)], pGPDp-GFP-2A-HIS3-K12(AAG) [K12(AAG)], or pGPDp-GFP-2A-HIS3-K12(AAG)FS [K12(AAG)FS] plasmid were analyzed as described in Figure 1C. (Right) The relative levels of each product normalized to the level of GFP-2A-His3 as 100% are shown as the mean values of three independent experiments with standard deviations.