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. 2007 Jan 26;35(3):999–1006. doi: 10.1093/nar/gkl1124

Figure 2.

Figure 2.

Hfq helps annealing of DsrA and rpoS. (A) Scheme to examine the annealing of DsrA and rpoSI mediated by Hfq. (B) Emission spectra of 25 nM Cy3-rpoSI and 50 nM Cy5-DsrA in T50 buffer without Hfq or after incubating the sample for 10 min without Hfq (black line), and then with 28 nM of Hfq (blue line). (B) Emission intensity time trace of Cy3 at 565 nm (green trace) and Cy5 at 667 nm (red trace). Hfq (28 nM) added at 10 min results in an abrupt increase (<20s) in FRET followed by a slow increase (average annealing time = 9 min). (D) EMSA experiment confirms the annealing reaction. Green and red bands correspond to the fluorescence scans with Cy3 and Cy5 filters respectively. Lane 1: Cy3-rpoSI (25 nM) and Cy5-DsrA (50 nM) in the absence of Hfq, Lanes 2–4: Cy3-rpoSI and Cy5-DsrA in the presence of Hfq (28 nM) after incubation for 5, 10 and 15 min at 15°C, Lane 5: Cy5-DsrA and Lane 6: Cy3-rpoSI. (E) The amount of Cy3-RNA in the annealed form (indicated in the gel image) is quantified and shown in the plot.