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RNA stores tau reversibly in complex coacervates

Fig 3

Tau tRNA binding by gel shift assay and isothermal titration calorimetry (ITC).

(A) Direct titration experiment shows 4R2N tau induces a mobility shift in tRNALys. To determine the Kd value, direct titration experiment was done, which requires trace RNA concentrations to meet the assumption used in the equation that derives the Kd value. The assumption is that the total protein concentration is approximately the free protein concentration at equilibrium, and that protein binding to RNA is negligible. In the direct titration experiments 26 nM, tRNA was used and protein concentration spans from 20 nM to 2 μM. (B) The fraction of bound tRNA by 4R2N tau plotted as a function of the monomeric tau concentration and fit to the Hill equation, y = 1 / [1 + (Kd / x)n]. (C) Yeast tRNA was titrated into solutions of 4R2N tau in an ITC experiment. The top panel shows the raw incremental-titration. The area under each peak is integrated and plotted against the tRNA:tau molar ratio and fitted to an independent binding model (the bottom panel), as discussed in Materials and methods. (B-C) SEM is reported from n = 3. (D) The stoichiometric binding experiments were performed by varying the tau:RNA molar ratio at a constant 2.6 μM concentration of tRNA, which approximates the saturation concentration (more than 5 times the Kd of 460 nM). The representative data of 3 independent experiments are shown. (E) Fraction of bound tRNA from the different bands in (D) is plotted over the molar tau:tRNA ratio. (F) Fraction of bound tau plotted as a function of tau:tRNA molar ratios and compared to the theoretical saturation binding curves (dotted lines) with protein:RNA molar stoichiometries of 1:1 to 6:1. The theoretical curves serve the purpose showing that multiple tau molecules bind tRNA with increasing tau concentrations, while the model is not meant to fit the data, given that multiple populations with different tau:tRNA ratios will coexist. The numerical data used in (B), (C), (E), and (F) are included in S1 Data.

Fig 3