Figure 3.

The RRMs as well as the RGG/RG motif contribute to the RNA chaperone activity of AUF1 p45. (a) (Top) Scheme of the structural rearrangement of the 5ʹ and 3ʹ termini, specifically of the 5ʹUAR and 3ʹUAR elements, during cyclization of the DENV RNA genome. (Bottom) Scheme of the fluorescence-based 3’SL^trunc-5ʹUAR interaction assay to detect AUF1 p45-mediated conformational rearrangement of DENV RNA by de-quenching of Cy5. (b) Example kinetic traces with Cy5 and BHQ (black hole quencher) labeled 3’SL^trunc incubated with the indicated concentrations of AUF1 p45 or variant RRM2_mut. Following the addition of 5ʹUAR RNA the fluorescence signals were measured, plotted as a function of time, and fitted according to a first-order reaction (no protein; Equation (1) or second-order reaction (in the presence of protein; Equation (2). (c) and (d) The observed rate constants k_obs (s⁻¹) that were measured for the RNA-RNA interaction reaction in the presence of AUF1 p45 or variants thereof were plotted as a function of the protein concentration.