FIGURE 6.
RNA-OUT interacts specifically with the proximal RNA-binding surface of Hfq. (A) EMSAs with 32P-labeled RNA-OUT (∼0.4 nM) and either WT or mutant forms of Hfq. HfqY25A is defective in RNA-binding at the distal site, and HfqK56A is defective in RNA-binding at the proximal site. The corresponding binding curves are presented below each gel image. Error bars represent standard error from two experiments. Note that all forms of Hfq used in this experiment possess a his6 epitope tag at their C termini. Species are labeled as in Figure 2. (B) EMSAs performed in the presence of competitor RNAs. HfqWT (untagged) was first mixed with various concentrations of DsrA or A18 RNA for 5 min, and 32P-labeled RNA-OUT (0.4 nM) was added. After an additional 15 min, reactions were subjected to polyacrylamide gel electrophoresis. A species expected to represent a ternary complex is labeled A18:Hfq:OUT*. IC50 values were calculated from curves shown in Supplemental Figure S4 and are reported in Table 2.