Figure 5. Reporter assay of avashort-bgaB WT and variants. (A) The putative anti-SD forming cytosines C34 and C35 were replaced by UA, UU, GA, and GG, respectively using randomized site-directed mutagenesis. The avashort WT exhibited a 3.5-fold induction of reporter activity after shift from 30–42 °C. All four variants exhibited WT-like activity. (B) Binding of AUG was loosened by changing the G55-C46 pair into a G55-A46 mismatch. Analyzed in the reporter assay, replacement of the cytosine by adenine resulted in complete destabilization of the avashort-RNAT structure visible in almost equal galactosidase activity levels at 30–42 °C. Both the results shown in (A and B) are mean values of a double measurement; mean standard deviation is given by error bars. WT, wild-type.