RNA recognition by minimal ProQ from Neisseria meningitidis

The 3′-terminal poly(U) tail is involved in rpmG-3′ and AniS-3′ binding to the N. meningitidis ProQ protein. (A) rpmG-3′ constructs with different lengths of 3′ poly(U) tails. (B) The fitting of the ProQ binding data using the quadratic equation provided K_d values of 0.1 nM for rpmG-U₆AU₂, 0.4 nM for rpmG-U₆AU, 1.2 nM for rpmG-3′, 0.5 nM for rpmG-U₅, and 0.4 nM for rpmG-U₄, while the binding for rpmG-U₂ reached saturation below 40% of bound RNA fraction, and rpmG-noU did not reach saturation up to 50 nM concentration of the ProQ. The K_d value for rpmG-U₆A was estimated as <0.5 nM. (C) AniS-3′ constructs with different lengths of 3′ poly(U) tails. (D) The fitting of the ProQ binding data using the quadratic equation provided K_d values of 4.9 nM for AniS-3′, 0.8 nM for AniS-U₇, 0.5 nM for AniS-U₆, 6.6 nM for AniS-U₅, and 1.1 nM for AniS-U₄, while the binding for AniS-U₂ did not reach saturation up to 50 nM concentration of the ProQ. The binding of AniS-noU was essentially undetectable up to 50 nM concentration of the ProQ. The data in the plots for rpmG-3′ and AniS-3′ binding to ProQ are the same as in Figure 1. The lower case g denotes guanosine residue added on 5′ end to enable T7 RNA polymerase transcription. Gels corresponding to the data in the plots are shown in Supplemental Figure S7. The RNA secondary structure predictions were performed in the ViennaRNA program (Lorenz et al. 2011). The average equilibrium dissociation constant (K_d) values and maximum RNA fraction bound calculated from at least three independent experiments are shown in Table 2.