Minimal binding assay of the DsrA–Hfq complex. 5′-end-labeled DsrA transcript (0.17 pmole) was subjected to limited alkaline hydrolysis to give ∼10% cleavage. The fragments of DsrA were incubated with purified Hfq protein, and bound and unbound fragments were subsequently separated on a native polyacrylamide gel. Bound and unbound DsrA fragments were excised, eluted, and fractionated on a denaturing polyacrylamide gel. Uncleaved DsrA (DsrA), 5′-end-labeled DsrADI (M), DsrA alkaline hydrolysis ladder (OH), DsrA fragments bound to Hfq (bound), and DsrA fragments that are not bound to Hfq (unbound) are represented in the figure. The bands seen in the bound lane at positions 25 and 35 are nonspecific and were likely due to small amounts of RNA degradation during purification of the DsrA bound fraction. These bands were not seen in other experiments
