Multiple native states reveal persistent ruggedness of an RNA folding landscape

Solomatin, Sergey V.; Greenfeld, Max; Chu, Steven; Herschlag, Daniel

doi:10.1038/nature08717

Cited by 203 publications

(248 citation statements)

References 31 publications

Supporting

Mentioning

237

Contrasting

Order By: Relevance

“…The lifetimes of both short-and long-lived dwells were similarly reduced in the A69G context. the group I intron and the hairpin ribozyme, dynamics of this kind have been attributed to conformational "memory" (39) or what has been more recently described as a "persistent ruggedness" of the RNA folding landscape (40). Such complexities imply "hidden" conformational changes in the molecule that have a direct impact on the observed signal.…”

Section: Resultsmentioning

confidence: 99%

Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution

Haller

Altman

Soulière

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

118

156

View full text Add to dashboard Cite

Thiamine pyrophosphate (TPP)-sensitive mRNA domains are the most prevalent riboswitches known. Despite intensive investigation, the complex ligand recognition and concomitant folding processes in the TPP riboswitch that culminate in the regulation of gene expression remain elusive. Here, we used single-molecule fluorescence resonance energy transfer imaging to probe the folding landscape of the TPP aptamer domain in the absence and presence of magnesium and TPP. To do so, distinct labeling patterns were used to sense the dynamics of the switch helix (P1) and the two sensor arms (P2/P3 and P4/P5) of the aptamer domain. The latter structural elements make interdomain tertiary contacts (L5/P3) that span a region immediately adjacent to the ligandbinding site. In each instance, conformational dynamics of the TPP riboswitch were influenced by ligand binding. The P1 switch helix, formed by the 5′ and 3′ ends of the aptamer domain, adopts a predominantly folded structure in the presence of Mg 2+ alone. However, even at saturating concentrations of Mg 2+ and TPP, the P1 helix, as well as distal regions surrounding the TPP-binding site, exhibit an unexpected degree of residual dynamics and disperse kinetic behaviors. Such plasticity results in a persistent exchange of the P3/P5 forearms between open and closed configurations that is likely to facilitate entry and exit of the TPP ligand. Correspondingly, we posit that such features of the TPP aptamer domain contribute directly to the mechanism of riboswitchmediated translational regulation.RNA | site-specific labeling | allosteric effect | structural preorganization | ergodicity R iboswitch elements located within noncoding regions of mRNA bind metabolites with high selectivity and specificity to mediate control of transcription, translation, or RNA processing (1, 2). In a manner that is controlled by metabolite concentration, nascent mRNAs containing riboswitch domains can enter one of two mutually exclusive folding pathways to impart regulatory control: the outcomes of these folding pathways correspond to ligand-bound or ligand-free states (3-5). These aptamer folds trigger structural cues into the adjoining expression platform, which, in turn, transduce a signal for "on" or "off" gene expression (6-9).The thiamine pyrophosphate (TPP)-sensing riboswitch is one of the earliest discovered regulatory elements in mRNA that is prevalent among bacteria, archaea, fungi, and plants (10-12). TPP riboswitches, sometimes present in tandem (13,14), control genes that are involved in the transport or synthesis of thiamine and its phosphorylated derivatives. The TPP-bound aptamer adopts a uniquely folded structure in which one sensor helix arm (P2/P3) forms an intercalation pocket for the pyrimidine moiety of TPP, and the other sensor helix arm (P4/P5) offers a water-lined binding pocket for the pyrophosphate moiety of TPP that engages bivalent metal ions (Fig. 1) (15-17). Like most riboswitch domains, structural information pertaining to the ligand-free TPP riboswitch is relatively ...

show abstract

Section: Resultsmentioning

confidence: 99%

Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution

Haller

Altman

Soulière

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

118

156

View full text Add to dashboard Cite

show abstract

“…9,10,16 Both combinations of ROXS-buffers (AA/MV or TX/TQ) could already prove their suitability to stabilize or alter the fluorescence properties of single fluorophores by photo-induced redox-reactions and are intensively used in single-molecule spectroscopy and super-resolution imaging. 9,10,16,29,30 The commercial AFs, namely Vectashield (VS) and Ibidi mounting medium (Ibidi-MM) contain phenylenediamine (PPD) and DABCO, respectively. In addition, these mounting media contain between 70 and 90% glycerol.…”

Section: Resultsmentioning

confidence: 99%

Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Cordes

Maiser

Steinhauer

et al. 2011

Phys. Chem. Chem. Phys.

View full text Add to dashboard Cite

Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore performance in fluorescence imaging of biological samples, we carried out photobleaching experiments using fixed cells labeled with various commonly used organic dyes, such as Alexa 488, Alexa 594, Alexa 647, Cy3B, ATTO 550, and ATTO 647N. Quantitative evaluation of (i) photostability, (ii) brightness, and (iii) storage stability of fluorophores in samples mounted in different antifades (AFs) reveal optimal combinations of dyes and AFs. Based on these results we provide guidance on which AF should preferably be used with a specific dye. Finally, we studied the antifading mechanisms of the commercial AFs using single-molecule spectroscopy and reveal that these empirically selected AFs exhibit similar properties to ROXS AFs.

show abstract

“…As a result, one needs the full free energy, enthalpy, and entropy landscapes for folding. A major road block in achieving a predictive understanding of RNA folding landscapes is that they are often "rugged,", i.e., with alternative conformations acting as kinetic traps (6,7). Moreover, the electrostatic challenge of folding a charged biopolymer highlights the particularly critical role of Mg 2þ and other counterions in the folding process.…”

mentioning

confidence: 99%

Entropic origin of Mg ²⁺ -facilitated RNA folding

Fiore

Holmstrom

Nesbitt

2012

Proc. Natl. Acad. Sci. U.S.A.

158

View full text Add to dashboard Cite

Mg 2þ is essential for the proper folding and function of RNA, though the effect of Mg 2þ concentration on the free energy, enthalpy, and entropy landscapes of RNA folding is unknown. This work exploits temperature-controlled single-molecule FRET methods to address the thermodynamics of RNA folding pathways by probing the intramolecular docking/undocking kinetics of the ubiquitous GAAA tetraloop−receptor tertiary interaction as a function of [Mg 2þ ]. These measurements yield the barrier and standard state enthalpies, entropies, and free energies for an RNA tertiary transition, in particular, revealing the thermodynamic origin of [Mg 2þ ]-facilitated folding. Surprisingly, these studies reveal that increasing [Mg 2þ ] promotes tetraloop-receptor interaction by reducing the entropic barrier (−T ΔS ‡ dock ) and the overall entropic penalty (−T ΔS°d ock ) for docking, with essentially negligible effects on both the activation enthalpy (ΔH ‡ dock ) and overall exothermicity (ΔH°d ock ). These observations contrast with the conventional notion that increasing [Mg 2þ ] facilitates folding by minimizing electrostatic repulsion of opposing RNA helices, which would incorrectly predict a decrease in ΔH ‡ dock and ΔH°d ock with [Mg 2þ ]. Instead we propose that higher [Mg 2þ ] can aid RNA folding by decreasing the entropic penalty of counterion uptake and by reducing disorder of the unfolded conformational ensemble.T he folding of RNA proceeds hierarchically, whereby secondary structure is formed rapidly and subsequent slow helical packing is mediated by tertiary interactions (1, 2). RNA secondary structure prediction from the known thermodynamics is quite reliable (3), though correspondingly accurate prediction of tertiary structure remains a major challenge (1). Static tertiary structure data alone are also not enough to predict RNA functionality, as time-dependent conformational dynamics occur during biochemical processes (4, 5). As a result, one needs the full free energy, enthalpy, and entropy landscapes for folding. A major road block in achieving a predictive understanding of RNA folding landscapes is that they are often "rugged,", i.e., with alternative conformations acting as kinetic traps (6, 7). Moreover, the electrostatic challenge of folding a charged biopolymer highlights the particularly critical role of Mg 2þ and other counterions in the folding process.Characterization of folding transition states-and the role of Mg 2þ in stabilizing transition states-remains a crucial bottleneck for reconciling the kinetics and thermodynamics of RNA folding (8-13). Some insight into the free energy landscapes for RNA folding can be obtained from temperature-dependent stopped-flow kinetic studies, which offer the ability to deconstruct free energy barriers (ΔG ‡ ) into enthalpic (ΔH ‡ ) and entropic (−TΔS ‡ ) components. However, with such methods, only the net rate constant (i.e., k total ¼ k fold þ k unfold ) for approach to equilibrium can be observed, which requires strong assumptions (e.g., that k fold ≫ k unfol...

show abstract

Multiple native states reveal persistent ruggedness of an RNA folding landscape

Cited by 203 publications

References 31 publications

Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution

Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution

Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Entropic origin of Mg ²⁺ -facilitated RNA folding

Contact Info

Product

Resources

About

Multiple native states reveal persistent ruggedness of an RNA folding landscape

Cited by 203 publications

References 31 publications

Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution

Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution

Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Entropic origin of Mg 2+ -facilitated RNA folding

Contact Info

Product

Resources

About

Entropic origin of Mg ²⁺ -facilitated RNA folding