Fig. 4. In vitro interactions of Hfq, polyP, and DNA.

(A) Two-dimensional analysis plots of frictional ratio f/f₀ versus sedimentation coefficient S for 25 μM Hfq ± 250 μM polyP₆₀ or polyP₃₀₀. The color in the z dimension represents the abundance of each species according to the partial concentration as indicated in the right y axis. (B) Mobility of Hfq (25 μM) on native gels without (lane 1) or with 65, 125, or 250 μM of various polyP chain lengths. PolyP₁₆ was used at 125 and 250 μM only (lanes 2 and 3). Gels were stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize polyP (cyan) followed by Coomassie staining to visualize Hfq (yellow). (C) Overlay of EMSA using FAM-labeled ybck-DNA (75 nM) in the presence of 30 μM Alexa Fluor 647 (AF647)–polyP₃₀₀ (lane 1); in the absence of additives (lane 2); or in the presence of 5, 10, 17.5, or 25 μM Hfq (lanes 3 to 6). AF647-polyP₃₀₀ (in Pi units) was added at 10, 15, 20, or 30 μM to 25 μM Hfq and DNA (lanes 7 to 10) or 25 μM Hfq only (11–15). Gels were stained with Coomassie to detect Hfq (fig. S4B).