FIGURE 5.

In vitro translation of the SAC51 5′ leader-GFP fusion transcript using a wheat-germ extract. (A) Structure of the three constructs used. The constructs of GFP alone (I), GFP fused with the SAC51 5′ leader (II), and GFP fused with the SAC51 5′ leader in which all start codons of uORFs are mutated (III), were transcribed in vitro using T7 RNA polymerase. (B) Effect of the amount of substrate transcripts on GFP synthesis. (C) Effect of thermospermine on GFP synthesis. 0.4 μg each of the in vitro transcript from the construct II was used in the translation reaction. (D) Effect of pretreatment by heat of the transcripts on GFP synthesis. 0.4 μg each of the in-vitro transcripts was treated at 25°C (Ct) or 65°C (Hs) for 10 min before translation.