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.

Characterizing RNA Excited States Using NMR Relaxation Dispersion

“…Many regulatory RNAs (Storz, 2002; Serganov et al, 2007; Cech et al, 2014) undergo conformational changes that play essential roles in their biological functions (Mandal et al, 2004; Tucker et al, 2005; Schwalbe et al, 2007; Cruz et al, 2009; Rinnenthal et al, 2011; Dethoff, Chugh, et al, 2012). Among many functionally important motional modes, changes in RNA secondary structure can expose or sequester key regulatory elements, and thereby provide the basis for molecular switches that regulate and control a wide range of biochemical processes (Serganov & Patel, 2007; Breaker, 2011; Dethoff, Chugh, et al, 2012).…”

Structural fidelity and NMR relaxation analysis in a prototype RNA hairpin

“…NMR is a major source of structural and dynamical information on nucleic acids in solution (Latham et al 2005;Fürtig et al 2007;Shajani and Varani 2007;Cruz and Westhof 2009;Rinnenthal et al 2011;Bardaro and Varani 2012;Salmon et al 2014), and is broadly supported by other forms of spectroscopy (Abdelkafi et al 1998;Leulliot et al 1999;Schiemann et al 2003;Qin and Dieckmann 2004;Bokinsky and Zhuang 2005;Zhuang 2005;Solomatin et al 2010). NMR-derived time averaged quantities such as dipolar couplings, chemical shifts, J-couplings, or NOEs can be translated into geometric restraints to refine RNA structures or conformational ensembles.…”

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

“…Further, it is unique in describing RNA dynamics at atomic resolution (Butcher et al 2000;Carlomagno et al 2013;Chowdhury et al 2006;D'Souza et al 2004;Davis et al 2005;Duszczyk et al 2011;Ferner et al 2009;Furtig et al 2003;HouckLoomis et al 2011;Keane et al 2015;Marchanka et al 2015;Sashital et al 2004;Staple and Butcher 2003;Wacker et al 2011). In fact, dynamics ranging from sub nanoseconds to seconds have been found to be essential to understand RNA function Rinnenthal et al 2011).…”