The functional Hfq-binding module of bacterial sRNAs consists of a double or single hairpin preceded by a U-rich sequence and followed by a 3′ poly(U) tail

Effects of mutations on Hfq binding in vivo. (A) Hfq binding of SgrS variants expressed from the low-copy plasmid. (B) Hfq binding of SgrS variants expressed from the high-copy plasmid. Crude extracts were prepared from TM771 (ΔsgrS hfq-FLAG-cat) cells harboring indicated plasmids and subjected to the pull-down assay using anti-Flag agarose as described in Materials and Methods. Crude extracts (CE; 5 μL) and bound fractions (B; 5 μL) were analyzed by Western blotting using anti-Flag antibodies. For analysis of RNAs, crude extracts (3 μL) and bound fractions (3 μL) were treated with phenol and subjected to Northern blotting using SgrS probe 1.