Figure 1.

The MS2-RsaA variant is normally expressed, correctly retained by affinity chromatography and functional. (A) Northern blots showing the expression of RsaA and the MS2-RsaA variant in HG001 WT and HG001-ΔrsaA strains. Total RNAs were prepared after 2, 4 and 6 h of culture in BHI medium at 37°C. Hybridization against 5S RNA was used as loading control. (B) Northern blot targeting RsaA, MS2-RsaA, mgrA or hu performed on RNAs purified after MS2 chromatography affinity; 1 μg of total RNA was loaded on a 2% agarose gel. CE: crude extract; FT : Flow-through ; W : Washing ; EL : Elution. mgrA* denotes a short fragment of the mgrA mRNA (below 274 nts) that was specifically detected by the mgrA probe in the elution fraction. This RNA fragment most likely represented a degradation product of mgrA mRNA containing the sequences interacting with RsaA. (C) In vitro translation assay performed with PURESYSTEM. The reactions were performed with 10 pmol of wild-type (WT) mgrA mRNA and in the presence of increasing quantities of WT RsaA or MS2-RsaA. The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (10%) and were revealed using a FLAG-specific antibody. *unspecific protein revealed with the FLAG-antibody, this protein was used as an internal loading control.