The 5′-terminal sequence preceding the terminator hairpin is involved in rpmG-3′ and AniS-3′ binding to the N. meningitidis ProQ protein. (A) rpmG-3′ constructs with different lengths of the 5′-terminal sequence. (B) The fitting of the ProQ binding data using the quadratic equation provided Kd values of 1.2 nM for rpmG-3′, 2.2 nM for rpmG-45, 0.6 nM for rpmG-35, and 7.0 nM for rpmG-33, while the binding for rpmG-31 did not reach saturation up to 50 nM concentration of the ProQ. The binding of rpmG-29 and rpmG-26 was essentially undetectable up to 50 nM concentration of the ProQ. (C) AniS-3′ constructs with different lengths of the 5′-terminal sequence. (D) The fitting of the ProQ binding data using the quadratic equation provided Kd values of 4.9 nM for AniS-3′, 5.0 nM for AniS-40, 4.5 nM for AniS-37, while the binding of AniS-35 was barely detected, and that of AniS-33, AniS-31, and AniS-29 was essentially undetected 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 S8. The RNA secondary structure predictions were performed in the ViennaRNA program (Lorenz et al. 2011). The average equilibrium dissociation constant (Kd) values and maximum RNA fraction bound calculated from at least three independent experiments are shown in Table 3.
