Real-Time Fluorescence-Based Approaches to Disentangle Mechanisms of a Protein's RNA Chaperone Activity.

Abstract

RNA-binding proteins with an RNA chaperone activity exert either one or both of the following catalytic activities: (1) RNA annealing, i.e., the protein supports intra- as well as intermolecular RNA-RNA interactions and (2) strand displacement, i.e., the protein mediates the exchange of individual strands of a preexisting RNA duplex. To discriminate and further characterize these activities, it requires defined assay systems. These are based on entirely or partially complementary RNA oligonucleotides that are labeled with fluorescent and/or quencher dyes. The non-catalyzed and the protein-supported associations of the RNA molecules are followed by a real-time fluorescence resonance energy transfer (FRET) system. By site-specific modification of the RNAs or the protein, the substrate- and protein-specific parameters of the RNA chaperone activity can be explored and identified.In this chapter, we present strategies on the design of labeled RNA molecules to be used to characterize the activities of an RNA-binding protein and explain how to monitor progress curves of RNA annealing and strand displacement reactions in single cuvette or well-plate scales. We provide sets of equations and models to determine and analyze different types of reactions, e.g., by calculation of first- and second-order rate constants. Likewise, we demonstrate how to exploit these simple experimental setups to elucidate elementary principles of the reaction mechanisms performed by the protein of interest by applying basic kinetic applications, such as ARRHENIUS and linear free energy relationship analyses. These approaches will be explained by providing example plots and graphs from experiments investigating the RNA chaperone activities of the RNA-binding proteins NF90-NF45 and AUF1 p45.