Thermodynamic analysis of ligand binding and ligand binding-induced tertiary structure formation by the thiamine pyrophosphate riboswitch

Kulshina, Nadia; Edwards, Thomas E.; Ferré-D′Amaré, A.R.

doi:10.1261/rna.1847310

Cited by 76 publications

(110 citation statements)

References 58 publications

(81 reference statements)

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“…This construct bound TPP with an estimated K d of about 100 nM (Fig. S2), comparable to the affinity reported for the unmodified aptamer (10,28,30).…”

Section: Resultssupporting

confidence: 79%

“…Conversely, the intrinsic instability of the native helix P1 sequence contained within the TPP aptamer domain investigated here (4 bp) may also contribute allosterically to the dynamics of the P4/P5 junction. For many previous folding and structural investigations of the TPP aptamer domain, helix P1 was either extended or altered (15,22,(24)(25)(26)(27)(28) to promote aptamer stability and compaction. In this view, the inherent instability of helix P1 may contribute directly to the mechanism of TPP riboswitchmediated translation.…”

Section: Resultsmentioning

confidence: 99%

“…Such studies include 2-aminopurine fold analysis (22), small-angle X-ray scattering (SAXS) (23)(24)(25), RNase-detected selective 2′-hydroxyl acylation (26,27), isothermal titration calorimetry (28,29), as well as single-molecule optical-trapping methods in which force was applied via the 5′ and 3′ ends of the RNA to directly monitor the energy landscape of TPP riboswitch folding and unfolding (30). Investigations of this kind have provided an important framework for understanding global features of the TPP riboswitch aptamer domain, revealing that its structural compaction is enabled by physiological concentrations of Mg 2+ ions and enforced by TPP binding.…”

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confidence: 99%

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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.

116

156

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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 ...

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“…This construct bound TPP with an estimated K d of about 100 nM (Fig. S2), comparable to the affinity reported for the unmodified aptamer (10,28,30).…”

Section: Resultssupporting

confidence: 79%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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.

116

156

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“…We then probed the TPP riboswitch RNA in the presence of subsaturating ligand concentrations [200 nM TTP; K d ∼50-200 nM (12)]. Clustering analysis of the chemical reactivity data produced three well-defined clusters in the ratio of 1:1:1.2 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule correlated chemical probing of RNA

Homan¹,

Favorov²,

Lavender³

et al. 2014

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

157

272

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Complex higher-order RNA structures play critical roles in all facets of gene expression; however, the through-space interaction networks that define tertiary structures and govern sampling of multiple conformations are poorly understood. Here we describe single-molecule RNA structure analysis in which multiple sites of chemical modification are identified in single RNA strands by massively parallel sequencing and then analyzed for correlated and clustered interactions. The strategy thus identifies RNA interaction groups by mutational profiling (RING-MaP) and makes possible two expansive applications. First, we identify throughspace interactions, create 3D models for RNAs spanning 80-265 nucleotides, and characterize broad classes of intramolecular interactions that stabilize RNA. Second, we distinguish distinct conformations in solution ensembles and reveal previously undetected hidden states and large-scale structural reconfigurations that occur in unfolded RNAs relative to native states. RING-MaP single-molecule nucleic acid structure interrogation enables concise and facile analysis of the global architectures and multiple conformations that govern function in RNA. These functions are mediated by tiered levels of information: The simplest is the primary sequence, and the most complex is the higher-order structure that governs interactions with ligands, proteins, and other RNAs (1, 2). Many RNAs can form more than one stable structure, and these distinct conformations often have different biological activities (3, 4). Currently, the rate of describing new RNA sequences vastly exceeds abilities to examine their structures.Here we characterize through-space interactions and multiple conformations in single RNAs by melding chemical probing and massively parallel sequencing. Because massively parallel sequencing reports the sequences of single templates, each read is fundamentally a single-molecule observation (5). We first modified RNA with a reagent that is sensitive to the underlying RNA structure and then detected multiple adducts in individual RNA strands (Fig. 1). Chemical adducts were detected as sequence mutations based on their ability to induce efficient misreading of the template nucleotide by a reverse transcriptase enzyme, an approach called mutational profiling, or MaP (6). Singlemolecule probing data were used in two distinct ways: to detect correlated RNA modifications reflecting higher-order through-space interactions (Fig. 1A) and to examine multiple conformations in single in-solution ensembles (Fig. 1B). Results and DiscussionMultisite Dimethyl Sulfate Reactivity with RNA. We used dimethyl sulfate (DMS) to probe the structures of three RNAs: the Escherichia coli thiamine pyrophosphate (TPP) riboswitch (79 nt) (7), the Tetrahymena group I intron P546 domain (160 nt) (8), and the Bacillus stearothermophilus RNase P catalytic domain (265 nt) (9). RNAs were selected to illustrate distinct RNA folding features and to emphasize increasingly difficult analysis challenges. The TPP riboswitch binds the...

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“…The metabolite-RNA complex inhibits translation by forming alternate structures and can discriminate between mono-and diphosphorylated analogs Mandal and Breaker 2004). The binding of a TPP metabolite also stabilizes additional tertiary interactions that are distant from the TPP binding sites (Serganov et al 2006;Lang et al 2007;Kulshina et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Thermodynamic examination of the pyrophosphate sensor helix in the thiamine pyrophosphate riboswitch

Furniss¹,

Grover²

2011

RNA

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Riboswitches are functional mRNA that control gene expression. Thiamine pyrophosphate (TPP) binds to thi-box riboswitch RNA and allosterically inhibits genes that code for proteins involved in the biosynthesis and transport of thiamine. Thiamine binding to the pyrimidine sensor helix and pyrophosphate binding to the pyrophosphate sensor helix cause changes in RNA conformation that regulate gene expression. Here we examine the thermodynamic properties of the internal loop of the pyrophosphate binding domain by comparing the wild-type construct (RNA WT) with six modified 2 3 2 bulged RNA and one 2 3 2 bulged DNA. The wild-type construct retains five conserved bases of the pyrophosphate sensor domain, two of which are in the 2 3 2 bulge (C65 and G66). The RNA WT construct was among the most stable (DG°3 7 = -7.7 kcal/mol) in 1 M KCl at pH 7.5. Breaking the A d G mismatch of the bulge decreases the stability of the construct~0.5-1 kcal/mol, but does not affect magnesium binding to the RNA WT. Guanine at position 48 is important for RNA-Mg 2+ interactions of the TPP-binding riboswitch at pH 7.5. In the presence of 9.5 mM magnesium at pH 5.5, the bulged RNA constructs gained an average of 1.1 kcal/ mol relative to 1 M salt. Formation of a single A+ d C mismatch base pair contributes about 0.5 kcal/mol at pH 5.5, whereas two tandem A+ d C mismatch base pairs together contribute about 2 kcal/mol.

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Thermodynamic analysis of ligand binding and ligand binding-induced tertiary structure formation by the thiamine pyrophosphate riboswitch

Cited by 76 publications

References 58 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

Single-molecule correlated chemical probing of RNA

Thermodynamic examination of the pyrophosphate sensor helix in the thiamine pyrophosphate riboswitch

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