Figure 7. Effect of mutations in RyhB-binding Sites I and II on msrB mRNA stability.

Northern blot analysis of wild type msrB (A), msrB mut1 (B), msrB mut2a (C), msrB mut1,2a (D), msrB mut2b (E), and msrB mut1,2b (F). Strains were grown at 37°C to an O.D.₆₀₀ of 0.4. After 10 min of incubation with 2,2′dip, rifampicin was added. Samples were removed at the times indicated after rifampicin addition and total RNA was extracted as described in Materials and Methods. Half-life of msrB mRNA was calculated with or without iron chelator. For determination of msrB mRNA amount, 10 μg of total RNA samples was loaded on a denaturating 1.2% agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for msrB and with 5S as a loading control. Half-life (seconds) of msrB mRNA (wild type and mutants) and the ratio of msrB mRNA half-life ±2,2′dip, are indicated. Band intensity of msrB transcript was normalized to that of 5S RNA.