Time-resolved NMR methods resolving ligand-induced RNA folding at atomic resolution

Abstract

spectroscopy ͉ riboswitches ͉ dynamics ͉ purine ͉ caged compound S tructural transitions of proteins and RNA constitute an important aspect of cellular function and information transfer. In proteins, the kinetics of these structural transitions, mainly from an unfolded ensemble to a single unique folded state, can be investigated by hydrogen exchange experiments monitored by NMR spectroscopy (1). Such studies have profoundly influenced our understanding of protein-folding pathways. In hydrogen exchange experiments, labile hydrogen atoms become protected against exchange with the bulk solvent during folding. This acquired exchange protection indicates formation of a persistent structure at atomic resolution. Incorporation of structural information derived from exchange experiments into molecular dynamics (MD) simulations (2), including data from methods exhibiting time and atomic resolution together with -value analysis (3) derived from mutational work, has provided detailed structural information of intermediates populated during folding.RNA molecules can adopt a variety of secondary and tertiary conformations (4, 5). In general, the energetic difference between alternate RNA conformations is very small, and the equilibrium distribution is strongly affected through the binding of proteins (6), ions (7), or small metabolites (8-10), or by structural modifications (11). Alternate RNA structures are stabilized by different Watson-Crick base-pairing interactions (12). To date, RNA folding has been investigated by using x-ray footprinting (13), oligonucleotide hybridization, and classical biochemical methods in conjunction with mutational data (14). Although hydrogen exchange rates can be used as reporters of RNA ground-state dynamics and stability (15-17), RNA hydrogen exchange experiments conducted in a pulse-chase manner fail in most cases because of the high intrinsic exchange rates observed even in folded RNA structures.Here, we report on ligand-induced conformational changes of an RNA at atomic resolution by using real-time NMR methods. We investigated the hypoxanthine-induced folding of the guanine-sensing riboswitch aptamer domain (GSR apt ) of the Bacillus subtilis xpt-pbuX operon (18). Riboswitch RNAs have emerged as an important example of macromolecular structural transitions that lead to transcriptional or translational regulation of protein expression induced through the specific binding of a metabolite (reviewed in refs. 19 and 20). Riboswitches are located in the 5Ј-untranslated region (5Ј-UTR) of bacterial mRNA and consist of a highly specific metabolite receptor region (aptamer domain) coupled to a 3Ј-downstream sequence (expression platform). Gene expression is thought to be modulated in response to conformational differences between the ligand-bound and ligand-free conformations of the aptamer domain (21). Crystal structures of the ligand-bound aptamer domains belonging to the class of purine riboswitches (guanineand adenine-sensing riboswitches) have revealed the presence of complex tertiary stru...

Loop–loop interaction in an adenine-sensing riboswitch: A molecular dynamics study

“…The structure of purine aptamer domains has been determined by X-ray crystallography (Batey et al 2004;Serganov et al 2004) and widely studied by NMR spectroscopy (Noeske et al 2005Buck et al 2007;Ottink et al 2007). It contains three conserved helical elements (P1, P2, and P3) that are present in all purine riboswitch sequences identified up until now.…”

Mechanisms for differentiation between cognate and near-cognate ligands by purine riboswitches

“…The global half-life for ligand association and complex formation is comparable to the xpt-aptamer:hypoxanthine complex at 2-fold ligand excess. However, for a 1:1 ratio of ligand to RNA, halflives of complex formation are ~10 sec for the mfl-aptamer and ~20-30 sec for the xpt-aptamer, 1,25 in agreement with less extensive structural re-arrangements of the aptamer core region during mfl-aptamer-2'-deoxyguanosine complex formation. In case of the mfl-aptamer, complex formation, the initial step of riboswitch-mediated gene regulation, is highly dependent on the correct identity of the ligand.…”

“…1,2 The ligand-free state is often substantially less structured and conformationally heterogeneous. However, preorganization of structural elements characteristic for the bound conformation in the absence of ligand has been observed, for example for the xpt-aptamer by NMR spectroscopy 3 or for the glmS-ribozyme by hydroxyl radical footprinting.…”

Three-way RNA junctions with remote tertiary contacts: A recurrent and highly versatile fold

“…In the sensing domain of TPP riboswitches, the bridging by the TPP ligand of the P2 and P3 internal loops likely induces an allosteric stabilization of the three-way junction via tertiary interactions that sequesters the switching strand contained within the P1 helix (Serganov et al 2006;Thore et al 2006). In purine riboswitches, the L2-L3 interaction is necessary to preorganize the junction for purine binding (Batey et al 2004;Lemay et al 2006;Buck et al 2007;Gilbert et al 2007;Noeske et al 2007;Ottink et al 2007;Greenleaf et al 2008;Stoddard et al 2008), and ligand binding at the junction also tightens the remote L2-L3 complex ). In hammerhead ribozymes, the L2-L3 interactions have been shown to reorganize the helical junction ( Fig.…”