Different RNA recognition by ProQ and FinO depends on the sequence surrounding intrinsic terminator hairpins

Comparison of malM-3′ and FinP RNA binding to ProQ, ProQ^NTD, and FinO. (A) Secondary structures of malM-3′ (black font) and FinP RNAs (red font), which were predicted using RNAstructure software (Reuter and Mathews 2010). The lowercase g denotes guanosine residue added on 5′ ends of RNA molecules to enable T7 RNA polymerase transcription. (B–D) The gelshift analysis of malM-3′ and FinP binding to full-length ProQ (B), ProQ^NTD (C), and FinO (D). Free ³²P-labeled RNA is marked as R and RNA-protein complexes as RP. (E) The plots of fraction-bound data versus protein concentration from B to D are shown. The fitting of the quadratic equation into malM-3′ binding data provided a K_d value of 18 nM for binding to ProQ, and 5.5 nM for binding to ProQ^NTD, while the K_d value for binding to FinO was estimated as higher than 200 nM. The fitting of the quadratic equation into FinP binding data provided a K_d value of 74 nM for binding to ProQ, 18 nM for binding to ProQ^NTD, and 109 nM for binding to FinO. The average equilibrium dissociation constant (K_d) values calculated from at least three independent experiments are shown in Table 1.