Figure 6. Varying the concentration of the 'trace' binding partner.

(A) Mixing scheme, as in Figure 4A but now with a series of labeled RNA concentrations. (B) Puf4 binding to different concentrations of ³²P-labeled RNA at 25°C. For simplicity, only the lower limits of RNA concentration are indicated; the corresponding upper limits were 15–140 pM RNA (see Materials and methods and Appendix 2—note 4). Incubation time t₁ was 0.5 hr, as established in Figure 4B. (C) Puf4 binding to different concentrations of ³²P-labeled RNA at 0°C. Lower limits of labeled RNA concentration are indicated. Incubation time t₁ was 40 hr. Note that these data are not fit well by Equation 4b, which assumes [R*]_total << K_D (solid lines). Quadratic fits, which do not assume negligible RNA concentration, are shown in dashed lines (Equation 5). (D) Effect of RNA concentration on apparent K_D (${\displaystyle K_{\text{D}}^{\text{app}}}$) at 0°C. Red symbols indicate ${\displaystyle K_{\text{D}}^{\text{app}}}$ values from a hyperbolic fit (Equation 4b and solid lines in C) and grey symbols indicate ${\displaystyle K_{\text{D}}^{\text{app}}}$ values from fits to the quadratic equation (Equation 5). The error bars denote 95% confidence intervals, as determined by fitting the data to the indicated equation in Prism 8.