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. 2015 Feb 10;43(4):2400–2411. doi: 10.1093/nar/gkv072

Figure 1.

Figure 1.

The A-rich fragments from the linker region of OxyS bind to the distal side of Hfq. (A) A schematic diagram showing the secondary structure of OxySU8. This structure is based on the experimentally verified secondary structure of OxyS (4), which is in close agreement with the predicted secondary structure of OxySU8 RNA using Mfold (43). The two segments selected for cocrystallization with Hfq, namely Aus and Ads, represent nucleotides 66–72 and nucleotides 68–74 of OxyS, respectively. OxySU8-A10dele (deletion of nucleotides 65–74) and OxySU8-A6U (A65U/A66U/A68U/A69U/A73U/A74U) mutants as indicated. Based on the Mfold prediction (43), the secondary structures of both mutants are similar to that of wild-type OxySU8. (B, C) Fluorescence polarization assay to determine the binding affinities of Aus and Ads for wild-type Hfq and mutants. A mutation on the distal side, Y25A, dramatically decreased the binding affinity of both Aus and Ads, whereas the proximal side mutation, F42S, did not exhibit a prominent effect. (D, E, F) TFQ experiments for Hfq Trp mutants by OxySU8, OxySU8-A10dele and OxySU8-A6U. F42W represents the proximal RNA binding site, and Y25W and K31W represent the distal RNA binding sites. The black bar represents the percent quenching by 1-μM RNA, whereas the gray bar above the black bar represents the quenching by 4-μM RNA.