25] Fluorescence assays to study structure, dynamics, and function of RNA and RNA-ligand complexes

Abstract

This chapter describes fluorescence assay for studying the structure, dynamics, and function of RNA and RNA–ligand complexes. The most abundant bases in RNA are guanine, adenosine, uracil, and cytosine. These natural bases do not fluoresce because of strong quenching in solution and cannot be used for fluorescence assays. This lack in autofluorescence enables the background-free use of site-specifically incorporated fluorophores as probes for their local environment. RNA from natural sources sometimes carries suitable intrinsic fluorophores, in particular the Wye (or Y) base. Intrinsic fluorescence from the protein component has been used to kinetically and thermodynamically characterize the formation of a number of protein–RNA complexes. Among them are complexes of aminoacyl-tRNA synthetases with their cognate transfer RNAs (tRNAs) of a translational repressor from T4 phage with the repressed mRNA, of both the nucleocapsid protein and the reverse transcriptase from human immunodeficiency virus (HIV) with their natural ligand tRNA(3Lys), or of the Rev protein from HIV with its RNA binding element.