FIGURE 6.

In vitro and in silico analysis of the secondary structure of the ptxS and PA5194 5′ UTRs. (A) Structural analysis of in vitro transcribed ptxS (left) and PA5194 (right) RNA with RNase T1 (T1), lead acetate (Pb²⁺) and RNase A (A) at different temperatures (indicated above the lanes in degrees Celsius). (-), ptxS or PA5194 RNA incubated at 37°C in the absence of RNases or lead acetate; (T) ptxS or PA5194 RNA digested with RNase T1 under denaturing conditions; (L) alkaline digestion of the PA5194 transcript. The dotted boxes encircle regions predicted to fold into secondary structures (shown in panel B) that involve the TIRs of the two RNA molecules. (B) Structure models of ptxS and PA5194 with probing results. Cleavage sites are indicated as full gray circles; in particular, positions showing thermo-dependent cleavage are indicated in dark gray. Stem–loops encompassing the SD and start codons of the two genes are enclosed in dotted boxes. The ptxS GUG start codon encompasses bases 78–80, the PA5194 AUG start codon bases 51–53. The three U's preceding the AUG in PA5194 that are mutated in pGM2013AAA and pGM2013CC are boxed. (C) Output of the RNAtips analysis of the two RNAs showing the density plot of positions along the ptxS (upper panel) or PA5194 (lower panel) sequences whose pairing probability is significantly affected by a temperature change from 28°C to 42°C. (D) Structure model of the E. coli lpxT TIR. The secondary structure was predicted with the software RNAfold. The start codon of the ORF is boxed; the black bar flanks the five uridine nucleotides involved in the pairing with the TIR. RNA secondary structures in B and D were drawn with the VARNA applet (Darty et al. 2009).