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-Δ rsaA 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, mgr A 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. mgr A* denotes a short fragment of the mgr A mRNA (below 274 nts) that was specifically detected by the mgr A probe in the elution fraction. This RNA fragment most likely represented a degradation product of mgr A 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) mgr A 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.