The gatekeeper of Yersinia type III secretion is under RNA thermometer control
Fig 5
Temperature-dependent melting of the SD sequence and start codon-containing stem-loop structure of the yopN RNAT.
(A) Enzymatic structure probing of the yopN RNAT (WT) and the stable variant (R1) at 25, 37 and 42°C. Radioactively labeled RNA was treated with single-strand specific RNases T1 (0.016 U) and T2 (0.025 U) at the different temperatures. LOH: alkaline Ladder; LT1: T1 treated RNA at 37°C; C: water control. (B) PARS-derived RNA secondary structure of the yopN RNAT [38]. White arrows indicate T1 and T2 cleavages at all temperatures while black arrows indicate cleavages with increasing temperature. The putative SD region is highlighted in gray and the start codon in red. (C) Quantification of band intensities at 25, 37 and 42°C of selected guanines and adenines. Pixel counting was performed using AlphaEaseFC software and values were normalized to intensities at 25°C. The enzymatic structure probing shown here represents one of two experiments performed.