FIG 6.

Effect of upstream mRNA stem-loop structure and A/U-rich sequence on translational activity and mRNA abundance. The translational activities of three different RBSs (RBS₁, RBS₂, and RBS₈) with and without upstream T7g10 mRNA stem-loop structure sequence (A), with and without upstream A/U-rich sequence (C), and with upstream A/U-rich sequences of different lengths (E) were evaluated by using the EYFP fluorescence reporter. To determine normalized absolute fluorescence, the optical density at 600 nm and EYFP fluorescence were measured. The mRNA abundance from constructs with and without upstream T7g10 mRNA stem-loop structure sequence (B), with and without upstream A/U-rich sequence (D), and with upstream A/U-rich sequences of different lengths (F) were measured by RT-PCR as described in Materials and Methods. Error bars represent standard deviations from three biological replicates. Asterisks indicate statistically significant increase in translational activity or eyfp mRNA abundance in the presence of stem-loop structure (A and B) or A/U-rich (C to F) sequences as follows: *, P < 0.01; **, P < 0.001; and ****, P < 0.00001 (unpaired t test).