Biophysical Characterization of the M_2-1 Transcription Antiterminator with RNA

The respiratory syncytial virus (RSV) encodes a singular transcription antiterminator or processivity factor M_2-1. This protein ensures the adequate expression of genes toward the 5' end of the genome that results from transcription polarization, in which genes located near the 3' genomic end are expressed at much higher levels than those at the 5' end, resulting in a gradient of transcripts. Although its mechanism of action is not fully understood, it is based on its RNA-binding capacity. This chapter describes biophysical spectroscopic methods for quantitatively analyzing the RNA binding activity of M_2-1 in vitro from the pure recombinant protein. By employing short fluorescently labeled RNA molecules, the chapter describes methodologies for characterizing physical interactions, determining binding stoichiometries and binding affinities, using a combination of electrophoretic mobility shift assays (EMSA) and equilibrium fluorescence spectroscopic titrations. In addition, the determination of dissociation constants, encompassing both independent single binding sites within the M_2-1 core domain and cooperative RNA binding to the M_2-1 tetramer, is described. Given the intricate nature and functional significance of RNA binding, the RSV M2-1 viral tetramer also constitutes a valuable model system for studying RNA-protein interactions among oligomeric proteins.