FIGURE 4.
Effect of upstream AUG (uAUG) on BTE-mediated translation. (A) Out-of-frame AUG (uAUG) was inserted upstream of the 5′-BCL. The arrows indicate the position of the uAUG and LUC AUG within nucleotides 74–177 of BYDV 5′-UTR. The underlined bases indicate the complementary bases forming the stem portion of the 5′-BCL, SL-D. The kissing bases are in the open box. The gray shading shows the sequence context of the upstream AUGs and the start codon of the luciferase gene. The uAUG construct has the same sequence context as the LUC AUG. In the Kozak uAUG construct, the uAUG is in the optimal context for initiation. The presence of the uAUG creates a small ORF (uORF) of 29 codons, which overlaps the 5′-end of the firefly luciferase coding region (fLUC). The amino acid residues encoded by the uORF and the 5′-end of the luciferase ORF are shown. (B) Relative luciferase activity of translation products of mRNAs containing the uAUG. (C) 35S-methionine-labeled in vitro translation products of indicated transcripts. Due to the presence of only one methionine residue in the uORF, the bottom panel was exposed at higher sensitivity using ImageQuant software to allow visualization of the uORF-encoded protein.