Paromomycin- and context-dependent stop codon read-through is recapitulated using the reporter for RNA cis-regulatory elements. (A) Effects of paromomycin on GFP fluorescence of strains with the TAA stop codon in good or poor contexts. Stop codons are flanked by sequences previously reported to cause low read-through (good context) or high read-through (poor context) (Bonetti et al. 1995). Scatter plots of GFP versus RFP fluorescence are shown in each set for a single strain grown in 0 μg/mL (red), 25 μg/mL (blue), and 100 μg/mL (orange) paromomycin. Strains on the left contain the insertion CAA-TAA-GCA beginning at codon 6 of GFP (good context), while strains on the right contain the sequence CAA-TAA-CAA at the same position (poor context). (B) Effects of paromomycin on median GFP/RFP levels from GFP constructs bearing stop codons in different sequence contexts. In the poor context, each stop codon is flanked by CAA on both its 5′ and 3′ side, while in the good context for TGA, the sequence is CAA-TGA-GAC, and for TAA, it is CAA-TAA-GCA. GFP/RFP medians were determined for four independent transformants of each construct at each concentration of paromomycin.
