Selective 2′-Hydroxyl Acylation Analyzed by Protection from Exoribonuclease

Steen, Kady Ann; Malhotra, Arun; Weeks, Kevin M.

doi:10.1021/ja103781u

Cited by 38 publications

(59 citation statements)

References 30 publications

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“…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.

115

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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Section: Resultsmentioning

confidence: 99%

mentioning

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.

115

156

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“…However, the authors did not demonstrate that this preorganization included any tertiary structure. Most recently, Steen, et al chemically modified the thiM RNA in the absence of ligand and observed a pattern of protections indicative of fully formed secondary structure, but no tertiary structure (23). Our data are consistent with all the data (but not necessarily all the interpretations) in these prior studies, as well as with the SAXS studies mentioned above, and argue that any preorganization of the A. thaliana thiC aptamer involves the formation of helices P1-P5 and the juxtaposition of those helices due to stacking or primary connectivity.…”

Section: Resultsmentioning

confidence: 99%

Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer

Anthony

Perez²,

Garcı́a-Garcı́a³

et al. 2012

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

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Riboswitches are motifs in the untranslated regions (UTRs) of RNA transcripts that sense metabolite levels and modulate the expression of the corresponding genes for metabolite import, export, synthesis, or degradation. All riboswitches contain an aptamer: an RNA structure that, upon binding ligand, folds to expose or sequester regulatory elements in the adjacent sequence through alternative nucleotide pairing. The coupling between ligand binding and aptamer folding is central to the regulatory mechanisms of thiamine pyrophosphate (TPP) riboswitches and has not been fully characterized. Here, we show that TPP aptamer folding can be decomposed into ligand-independent and -dependent steps that correspond to the formation of secondary and tertiary structures, respectively. We reconstructed the full energy landscape for folding of the wild-type (WT) aptamer and measured perturbations of this landscape arising from mutations or ligand binding. We show that TPP binding proceeds in two steps, from a weakly to a strongly bound state. Our data imply a hierarchical folding sequence, and provide a framework for understanding molecular mechanism throughout the TPP riboswitch family. Riboswitches that sense the essential coenzyme thiamine pyrophosphate (TPP) are found in all kingdoms of life, and regulate thiamine synthesis at the level of transcription, translation, or splicing (1-3). Members of the TPP riboswitch family share sequence elements, architectures, and modes of ligand binding (4-9). The TPP-binding aptamer in the 3′ UTR of the thiC gene from Arabidopsis thaliana possesses a "tuning-fork" architecture (10) comprising two sensor helix arms (P2∕3 and P4∕5) and a switch helix (P1), all stemming from a central junction (J2∕4) (Fig. 1A). The aptamer is thought to bind its ligand as the sensor helix arms are brought together, and bulges (J2∕3 and J4∕5) in the arms join to form a bipartite binding pocket. Purine riboswitches also resemble tuning forks (11, 12), but in contrast have a single binding pocket comprised largely of nucleotides from the central junction.It has been proposed that riboswitches may be sorted into two functional types (13): Type I and Type II, of which the purine and TPP riboswitches are prototypic examples, respectively [as more riboswitch sequences and structures have been determined, additional classification schemes have been discussed (1, 14)]. The two types are distinguished by binding pocket architecture and the scale of the structural rearrangement accompanying ligand binding, with Type I and Type II undergoing local and longdistance rearrangements, respectively. An earlier single-molecule study (15) of the Type I pbuE aptamer from Bacillus subtilis, which binds adenine, revealed that secondary and tertiary structure formation were interleaved during folding, in that a competent binding site (constituting a tertiary element) was formed prior to the closure of the base of the switch helix, P1 (a secondary element). Here, we have extended the single-molecule approaches used previously to in...

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“…28,32 In another example, SHAPE analysis of the free and unbound thiamine pyrophosphate sensing (TPP) riboswitch aptamer emphazised that, in the ligand-free state, the aptamer consists of five well-formed and stable helices that are linked by highly dynamic singlestranded regions, suggestive of a structure with little or no stable tertiary interactions. 33 Noteworthily, hydroxyl radical footprinting and UV-cross-linking have also been alternatively applied to verify preorganized states of the glmS riboswitch in the absence of ligand. 34 While biochemical probing techniques can provide a wealth of information on nucleotides and sequence stretches of an unliganded riboswitch that interact with each other, they yield limited information about the overall architecture and, in particular, the interhelical organization.…”

Section: What Experimental Methods Grant Insights Into the Free Ribosmentioning

confidence: 99%

The Dynamic Nature of RNA as Key to Understanding Riboswitch Mechanisms

2011

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Riboswitches are gene regulation elements within RNA that recognize specific metabolites. They predominantly occur in the untranslated leader regions of bacterial messenger RNA (mRNA). Upon metabolite binding to the aptamer domain, a structural change in the adjoining downstream expression platform signals "on" or "off" for gene expression. Researchers have achieved much progress in characterizing ligand-bound riboswitch states at the molecular level; an impressive number of high-resolution structures of aptamer-ligand complexes is now available. These structures have significantly contributed toward our understanding of how riboswitches interact with their natural ligands and with structurally related analogues. In contrast, relatively little is known about the nature of the unbound (apo) form of riboswitches. Moreover, the details of how changes in the aptamer domain are transduced into conformational changes in the decision-making expression platform remain murky. In this Account, we report on recent efforts aimed at the characterization of free states, ligand recognition, and ligand-induced folding in riboswitches. Riboswitch action is best approached as a cotranscriptional process, which implies sequential folding and release of the aptamer prior to the signaling of the expression platform. Thus, a complex interplay of several factors has to be taken into account, such as speed of transcription, transcriptional pausing, kinetics and thermodynamics of RNA structure formation, and kinetics and thermodynamics of ligand binding. The response mechanism appears to be best described as a process in which ligand recognition critically dictates the folding pathway of the nascent mRNA during its expression; the resulting structures determine the interactions with the transcriptional or translational apparatus. We discuss experimental methods that offer insight into the dynamics of the free riboswitch state. These include probing experiments, such as in-line and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) techniques, small-angle X-ray scattering (SAXS) analysis, NMR spectroscopy, and fluorescence spectroscopy, including single-molecule fluorescence resonance energy transfer (smFRET) imaging. One of our research contributions is an approach, termed 2ApFold, that incorporates noninvasive 2-aminopurine modifications in riboswitches. The fluorescence response of these moieties is used to delineate the order of secondary-tertiary structure formation and rearrangements taking place during ligand-induced folding. This information can be used to explore the kinetics of ligand recognition and to analyze the degree of structure preorganization of the free riboswitch state. Furthermore, we discuss a recent smFRET study on the SAM-II riboswitch; this report underscores the importance of choosing strategic labeling patterns that leave maximal conformational freedom to the regulatory interaction. Finally, we comment on how riboswitch ligand recognition appeals to the concepts of conformational selection ...

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Selective 2′-Hydroxyl Acylation Analyzed by Protection from Exoribonuclease

Cited by 38 publications

References 30 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

Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer

The Dynamic Nature of RNA as Key to Understanding Riboswitch Mechanisms

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