Figure 4.

The length of the uORF determines translation reinitiation efficiency. (A) Extension of the uORFs in A1 and A2 constructs. The A1 and A2 uAUGs were fused to the A5 uAUG, thereby creating constructs A1–A5 and A2–A5 with an uORF size of 93 nt. The U1–U5 and U2–U5 were created similarly with the U1, U2 and U5 constructs, respectively. In addition, the uORF in A2 was extended in A2-LF by insertion of a G at nt +123 (shown by an asterisk), which destroys the original stop codon. As a consequence, the uORF length is extended from 18 to 150 nt, and the intercistronic distance is decreased by 132 nt. (B and C) Transfections were performed as in Figure 3A and B. One of three independent experiments is shown. Error bars indicate standard deviation. (D and E) Western blot analysis of the luciferase proteins. See Figure 3C and D for details.