Effects of distance between base-pairing region and Hfq-binding module. (A) Nucleotide sequences of SgrS-S and the spacing mutants. The inserted sequences are derived from the gfp gene. (B) Properties of SgrS-S variants. IT1568 cells harboring indicated plasmids were grown in the presence of 0.2% arabinose. Total RNAs and proteins were prepared. RNA samples were resolved by 10% polyacrylamide gel electrophoresis in the presence of 8 M urea and subjected to Northern blot analysis using SgrS probe 2 and a ptsG probe. The following amounts of RNAs were loaded: SgrS variants, 15 μg; ptsG mRNA, 15 μg. Protein samples equivalent to 0.04 A600 units were subjected to Western blot analysis. (C) Hfq binding of SgrS-S variants. Crude extracts (CE) and bound fraction (B) were prepared from TM771 (ΔsgrS hfq-FLAG-cat) cells harboring indicated plasmids as described in Materials and Methods. The crude extracts (200 μL) and the bound fraction (20 μL) were treated with phenol, precipitated, and washed with ethanol. Each precipitate was dissolved in 10 μL of RNA buffer (0.02 M sodium acetate at pH 5.5, 0.5% SDS, and 1 mM EDTA), resolved by 10% polyacrylamide gel electrophoresis in the presence of 8 M urea and subjected to Northern blotting using SgrS probe 2.
