Figure 2.

Flow cytometry analysis of constructs with one uAUG in the most optimal context and a longer leader of 94 nt and constructs containing a stem–loop near the 5′-cap site in transiently transfected mammalian cells. (A) Schematic showing constructs containing one uAUG in the most optimal context with a short leader of 20 nt (uATG_b), a leader of 94 nt (uATG_bL) or containing a stem–loop 14 nt downstream of the 5′-cap site (uAUG_bL-SL). The control constructs lacking uAUGs, with (pEGFP-N1-SL) and without (pEGFP-N1) stem–loops, are shown. The various leader lengths between the 5′-cap and first AUG in these constructs are indicated by arrows. The uAUG is underlined and the beginning of the GFP ORF is indicated by a bent arrow. The sequence between the uAUG and main AUG is shown in the insert box. (B) GFP fluorescence intensity histograms compiled from the analysis of 20 000 cells per sample for each construct in transfected HeLa cells. It shows that construct uATG_bL expresses GFP fluorescence levels that are similar to construct uATG_b. Both stem–loop constructs (uAUG_bL-SL and pEGFP-N1-SL) display GFP fluorescence levels that are similar to the untransfected control. (C) Graphical representation of GFP intensities equating to translation efficiency for each construct in transfected HeLa cells.