FIG 1.

The 5′ and 3′ borders of the TriMV 5′ UTR are necessary for the TriMV IRES activity. (A) Schematic diagram of the bicistronic dual-luciferase reporter RNA and the positions of the insertions of the RNA element tested. Translation of the renilla luciferase gene is cap mediated, but translation of the downstream firefly luciferase gene can be directed only by internal initiation driven by the RNA sequence inserted into the intergenic region. The internal initiation ability is quantified as the ratio of firefly luciferase to renilla luciferase activities. The ratio of firefly to renilla luciferase activities in wheat germ extract of the bicistronic mRNAs was determined by a m7 GpppG cap at the 5′ end and the TriMV 5′ UTR (construct 1-739) or the nonfunctional TriMV reverse complementary sequence (construct 739-1) or the deletion mutants in the intergenic region. (B) Schematic diagram of monocistronic firefly luciferase reporter RNA containing a stable hairpin insertion (ΔG = –34 kcal) immediately at the 5′ end of the mRNA (12). The relative luciferase activities in wheat germ extract of the TriMV 5′ UTR deletion mutants with the strong hairpin is shown and is relativized to that of the TriMV wild-type sequence. (C) Relative luciferase activity in oat protoplasts of the reporter mRNAs containing the full-length TriMV leader (construct 1-739 SL) or the deletion mutant missing the last 30 nt (construct 1-709 SL) as 5′ UTR with the strong hairpin, normalized to values for a m7GpppG-capped polyadenylated renilla mRNA used as an internal control, which we coelectroporated at a 1:10 ratio. As a control, we included the full-length TriMV leader sequence as 5′ UTR in the absence of the strong stem-loop (construct 1-739 noSL) and the nonfunctional TriMV reverse complementary sequence (construct 739-1 SL).