Figure 7. Reporter gene assay with avalong-RNAT WT and variants. (A) Using site-directed mutagenesis, the terminal H1-loop (Nt 31–70), the bulged uridine 90 in the ROSE-like anti-SD was deleted (ΔU90) and two flanking GC pairs were opened by exchanging the cytosines at positions 88 and 91 against adenines (CC88/91AA). Compared with the WT exhibiting a 10-fold increase after heat shock from 30–42 °C, deletion of the top-loop resulted in high β-galactosidase activity at 30 °C and decreased heat-induction after the shift to 42 °C. In contrast, deletion of the predicted bulged U90 provoked low levels of reporter activity both under heat shock and non-heat shock conditions. A loosened structure around the SD led to a high reporter gene expression at 30 °C and 42 °C. The results reflect mean values of a double-measurement; mean standard deviation is given by error bars. (B) Measurement of β-galactosidase activity at 30 °C and 42 °C of avalong-variants harboring a mutated alternative anti-SD sequence. Neither the substitution of C53, C54 by adenines (avalong_*2A), nor of the CCUC motif (Nt 53–56, avalong_*4A) led to a derepression at low temperatures compared with the wild-type.