Fluorescence tools to investigate riboswitch structural dynamics

St-Pierre, Patrick; McCluskey, Kaley; Shaw, Euan; Penedo, J. Carlos; Lafontaine, Daniel A.

doi:10.1016/j.bbagrm.2014.05.015

Cited by 27 publications

(23 citation statements)

References 95 publications

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“…The difficulty arises in trying to define often short-lived intermediates that are very difficult to identify and quantify. Previous attempts have utilized a range of time-resolved techniques such as footprinting (Sclavi et al 1998;Silverman et al 2000;Shcherbakova et al 2004), scattering (both X-ray and neutron) (Russell et al 2000;Das et al 2003;Perez-Salas et al 2004;Moghaddam et al 2009;Roh et al 2010;Pollack 2011), as well as fluorescence Haller et al 2011;Buskiewicz and Burke 2012;St-Pierre et al 2014;Frener and Micura 2016). Our experiments directly observe continuous changes in the orientations and stacking of the 2AP nucleobases (local events) as well as changes in the global conformational ensemble observed via SAXS during the transition from secondary structure to tertiary folds.…”

mentioning

confidence: 73%

Divalent ions tune the kinetics of a bacterial GTPase center rRNA folding transition from secondary to tertiary structure

Welty

Pabit

Katz

et al. 2018

RNA

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Folding of an RNA from secondary to tertiary structure often depends on divalent ions for efficient electrostatic charge screening (nonspecific association) or binding (specific association). To measure how different divalent cations modify folding kinetics of the 60 nucleotide E. coli rRNA GTPase center, we combined stopped-flow fluorescence in the presence of Mg 2+ , Ca 2+ , or Sr 2+ together with time-resolved small angle X-ray scattering (SAXS) in the presence of Mg 2+ to observe the folding process. Immediately upon addition of each divalent ion, the RNA undergoes a transition from an extended state with secondary structure to a more compact structure. Subsequently, specific divalent ions modulate populations of intermediates in conformational ensembles along the folding pathway with transition times longer than 10 msec. Rate constants for the five folding transitions act on timescales from submillisecond to tens of seconds. The sensitivity of RNA tertiary structure to divalent cation identity affects all but the fastest events in RNA folding, and allowed us to identify those states that prefer Mg 2+ . The GTPase center RNA appears to have optimized its folding trajectory to specifically utilize this most abundant intracellular divalent ion.

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mentioning

confidence: 73%

Divalent ions tune the kinetics of a bacterial GTPase center rRNA folding transition from secondary to tertiary structure

Welty

Pabit

Katz

et al. 2018

RNA

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“…In the past decade, development of sophisticated biophysical techniques with significantly improved sensitivity, including single molecule [17–19], time-resolved hydroxyl radical footprinting [20], and fluorescence techniques [21], have allowed for discovery and characterization of novel excited conformational states involved in both RNA folding and recognition of proteins and ligands at single-molecule and single-nucleotide resolution. However, accurate assignment of a specific molecular configuration to an excited state remains a challenge for these low-resolution experimental approaches.…”

Section: Introductionmentioning

confidence: 99%

Characterizing excited conformational states of RNA by NMR spectroscopy

Zhao

Zhang

2015

Current Opinion in Structural Biology

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Conformational dynamics is a hallmark of diverse non-coding RNA functions. During these functional processes, RNA molecules almost ubiquitously undergo conformational transitions that are tuned to meet distinct structural and kinetic requirements for proper function. A complete mechanistic understanding of RNA function requires comprehensive structural and dynamic knowledge of these complex transitions, which often involve alternative higher-energy conformational states that pose a major challenge for high-resolution structural study by conventional methods. In this review, we describe recent progress in RNA NMR that has started to unveil detailed structural, thermodynamic and kinetic insights into some of these excited conformational states of RNA and their functional roles in biology.

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“…However, due to the difficulties in probing dynamic and lowly populated conformations using such ensemble methods, ligand-free conformations are often recalcitrant to structural inquiry. Consequently, single molecule fluorescence resonance energy transfer (smFRET) has become an increasingly popular tool to avoid ensemble averaging and study ligand-free riboswitch conformations in solution, as well as their dynamics and ligand dependent folding pathways (Liberman et al, 2012; Savinov, Perez & Block, 2014; St-Pierre, McCluskey, Shaw, Penedo & Lafontaine, 2014; Suddala et al, 2013). …”

Section: Introductionmentioning

confidence: 99%

Riboswitch Structure and Dynamics by smFRET Microscopy

Suddala

Walter

2014

Methods in Enzymology

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Riboswitches are structured non-coding RNA elements that control the expression of their embedding messenger RNAs by sensing the intracellular concentration of diverse metabolites. As the name suggests, riboswitches are dynamic in nature so that studying their inherent conformational dynamics and ligand-mediated folding is important for understanding their mechanism of action. Single molecule fluorescence energy transfer (smFRET) microscopy is a powerful and versatile technique for studying the folding pathways and intra- and intermolecular dynamics of biological macromolecules, especially RNA. The ability of smFRET to monitor intramolecular distances and their temporal evolution make it a particularly insightful tool for probing the structure and dynamics of riboswitches. Here, we detail the general steps for using prism-based total internal reflection fluorescence (TIRF) microscopy for smFRET studies of the structure, dynamics and ligand binding mechanisms of riboswitches.

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Fluorescence tools to investigate riboswitch structural dynamics

Cited by 27 publications

References 95 publications

Divalent ions tune the kinetics of a bacterial GTPase center rRNA folding transition from secondary to tertiary structure

Divalent ions tune the kinetics of a bacterial GTPase center rRNA folding transition from secondary to tertiary structure

Characterizing excited conformational states of RNA by NMR spectroscopy

Riboswitch Structure and Dynamics by smFRET Microscopy

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