Chapter 11 Single-Molecule Fluorescence Methods for the Analysis of RNA Folding and Ribonucleoprotein Assembly

Abstract

Publisher Summary In addition to its role as an information carrier in gene expression, RNA is now recognized to carry out a broad range of biological functions, and novel activities continue to emerge on a regular basis. RNA molecules can act as catalysts in reversible phosphodiester-cleavage reactions, mediate splicing of premessenger RNA, silence specific genes, and act as molecular switches to sense metabolites and regulate the translation of genes into proteins, to name just a few important examples. Perhaps the most impressive (and unexpected) function of RNA is the ability to catalyze peptide bond formation on the ribosome. Similar to proteins, RNAs are synthesized as linear polymers that must fold into compact three-dimensional structures to attain their biological activity. However, there are fundamental differences between these two types of macromolecular folding processes. While small RNAs, including many ribozymes, can fold autonomously, most large RNA molecules require one or more protein chaperones for efficient and correct folding. Proteins can prevent misfolding of large RNAs and/or accelerate the escape from kinetic traps. Additionally, proteins may guide proper folding by manipulating the structure of the RNA chain. Chemical cross-linking can capture RNA folding intermediates, but the technique provides only limited structural information. Fluorescence spectroscopy can provide both structural and dynamic information and is emerging as a powerful tool for studies of RNA folding and RNP assembly processes. Fluorescence measurements can be performed in solution under physiologically relevant conditions, without restrictions arising from the size of the molecules under study. Moreover, the method provides dynamic information spanning a wide range of time scales, from picoseconds to minutes.