Figure 6.

Double base substitutions in either side of the pseudoknot stem 2 impair toxicity, while compensatory mutants are lethal in vivo. (A) (Left) Schematics of the pseudoknot, for comparison. (Right) Boxes show stem 2 only, using the same color code as on the left. Double nucleotide substitutions on each arm, and compensatory quadruple mutations (purple) are indicated. (B) E. coli cells carrying an empty vector control (ct) or pBAD plasmids expressing the wild-type +42 tisB mRNA, or mRNAs with the U56C/G57C, C74G/A75G or Comp. 2 mutation, were grown to OD₆₀₀ = 0.3 in a Tecan plate reader before addition (red arrow) of 0.025% glucose (left) or arabinose (right). All experiments in B, D, F were conducted four times. (C) Ten-fold serial dilutions of bacterial cultures (same strains as in B) were subjected to plating assays as in Figure 5C. (D, E). Same as (B, C) but with cells carrying pBAD plasmids expressing U56C/G57C, C74G/A75G, or Comp. 3 +42 tisB mRNAs. (F, G). Same as (B, C) but with cells carrying pBAD plasmids expressing G54/G57, C74G/C77G, or Comp. 4 +42 tisB mRNAs.