FIGURE 3.

Two noncomplementary short RNAs bind simultaneously to Hfq or dimerization-competent wild-type StpA. (A) Monitoring “RNA crowding.” Two noncomplementary fluorophore-labeled RNAs give a FRET signal when they are in close proximity because of simultaneous binding to a protein. In this model, dual RNA binding is mediated by two StpA proteins that dimerize via their N-terminal domain. (B) In a microplate reader, the noncomplementary RNAs Cy5–21R+ and Cy3-Duplex− were injected into buffer containing the respective proteins. The FRET index was measured and calculated as described in the Materials and Methods but not normalized. Only incubation with StpA and Hfq yielded quantifiable reaction curves; the observed rate constants for dual binding k _db are 0.028±0.002 sec⁻¹ and 0.027±0.002 sec⁻¹.