Mapping the landscape of RNA dynamics with NMR spectroscopy

Abstract

Among the three major classes of biomacromolecules (DNA, RNA, and proteins) RNA's pronounced dynamics are the most explicitly linked to its wide variety of functions, which include catalysis and the regulation of transcription, translation, and splicing. These functions are mediated by a range of RNA biomachinery, including such varied examples as macromolecular noncoding RNAs, microRNAs, small interfering RNAs, riboswitch RNAs, and RNA thermometers. In each case, the functional dynamics of an interconversion is characterized by an associated rate constant. In this Account, we provide an introduction to NMR spectroscopic characterization of the landscape of RNA dynamics. We introduce strategies for measuring NMR parameters at various time scales as well as the underlying models for describing the corresponding rate constants. RNA exhibits significant dynamic motion, which can be modulated by (i) intermolecular interactions, including specific and nonspecific binding of ions (such as Mg(2+) and tertiary amines), (ii) metabolites in riboswitches or RNA aptamers, and (iii) macromolecular interactions within ribonucleic protein particles, including the ribosome and the spliceosome. Our understanding of the nature of these dynamic changes in RNA targets is now being incorporated into RNA-specific approaches in the design of RNA inhibitors. Interactions of RNA with proteins, other RNAs, or small molecules often occur through binding mechanisms that follow an induced fit mechanism or a conformational selection mechanism, in which one of several populated RNA conformations is selected through ligand binding. The extent of functional dynamics, including the kinetic formation of a specific RNA tertiary fold, is dependent on the messenger RNA (mRNA) chain length. Thus, during de novo synthesis of mRNA, both in prokaryotes and eukaryotes, nascent mRNA of various lengths will adopt different secondary and tertiary structures. The speed of transcription has a critical influence on the functional dynamics of the RNA being synthesized. In addition to modulating the local dynamics of a conformational RNA ensemble, a given RNA sequence may adopt more than one global, three-dimensional structure. RNA modification is one way to select among these alternative structures, which are often characterized by nearly equal stability, but with high energy barriers for conformational interconversion. The refolding of different secondary and tertiary structures has been found to be a major regulatory mechanism for transcription and translation. These conformational transitions can be characterized with NMR spectroscopy, for any given RNA sequence, in response to external stimuli.