Allosteric Tertiary Interactions Preorganize the c-di-GMP Riboswitch and Accelerate Ligand Binding

Wood, Sharla; Ferré-D′Amaré, A.R.; Rueda, David

doi:10.1021/cb300014u

Cited by 62 publications

(93 citation statements)

References 55 publications

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“…3). We hypothesize that such behaviors arise from relatively slow conformational changes [also observed in other riboswitch systems (19,33)] within the core domain of the TPP aptamer within the J2-4 junction and related distortions in the RNA backbone at the union of the P4 and P5 helices (Fig. 1).…”

Section: Resultsmentioning

confidence: 77%

“…Finally, the dynamic behavior of the TPP riboswitch is surprisingly distinct from other junctional riboswitches, such as purine and c-di-GMP riboswitches previously investigated by smFRET methods (32,33,47). In those systems, long-range tertiary interactions formed between remote parts of the molecule distant from the actual ligand-binding sites were shown to be stably formed to efficiently interact with the ligand and, in turn, to allow for a mutually exclusive gene response.…”

Section: Resultsmentioning

confidence: 89%

“…However, it is not yet clear how the collapse of the two helical domains and the formation of these tertiary interactions are influenced by ligand binding or whether they are essential to binding pocket formation. Here, we have used single-molecule fluorescence resonance energy transfer (smFRET) (31)(32)(33) imaging to track ligand-dependent changes in the TPP riboswitch from multiple structural perspectives to elucidate the relationship between TPP recognition and aptamer folding.…”

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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: 77%

Section: Resultsmentioning

confidence: 89%

mentioning

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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“…Detailed smFRET studies have been recently performed on a number of RNA systems, including the c-di-GMP, SAM-I, and lysine riboswitches, to elucidate the dynamics underlying the riboswitch folding process. In the case of the c-di-GMP riboswitch (48), such studies revealed that the free RNA occupies four distinct configurations that appeared to differ dramatically in their dynamic properties and apparent stabilities. Here, evidence was presented that all four states interconvert over very long (ca.…”

Section: Large Structurally Complex Riboswitches With Pseudoknots Andmentioning

confidence: 99%

Tuning a riboswitch response through structural extension of a pseudoknot

Soulière

Altman

Schwarz

et al. 2013

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

102

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Structural and dynamic features of RNA folding landscapes represent critical aspects of RNA function in the cell and are particularly central to riboswitch-mediated control of gene expression. Here, using single-molecule fluorescence energy transfer imaging, we explore the folding dynamics of the preQ 1 class II riboswitch, an upstream mRNA element that regulates downstream encoded modification enzymes of queuosine biosynthesis. For reasons that are not presently understood, the classical pseudoknot fold of this system harbors an extra stem-loop structure within its 3′-terminal region immediately upstream of the Shine-Dalgarno sequence that contributes to formation of the ligand-bound state. By imaging ligand-dependent preQ 1 riboswitch folding from multiple structural perspectives, we reveal that the extra stem-loop strongly influences pseudoknot dynamics in a manner that decreases its propensity to spontaneously fold and increases its responsiveness to ligand binding. We conclude that the extra stem-loop sensitizes this RNA to broaden the dynamic range of the ON/OFF regulatory switch.SHAPE | single-molecule FRET | site-specific labeling | metabolite sensing RNAs | ligand recognition A variety of small metabolites have been found to regulate gene expression in bacteria, fungi, and plants via direct interactions with distinct mRNA folds (1-4). In this form of regulation, the target mRNA typically undergoes a structural change in response to metabolite binding (5-9). These mRNA elements have thus been termed "riboswitches" and generally include both a metabolite-sensitive aptamer subdomain and an expression platform. For riboswitches that regulate the process of translation, the expression platform minimally consists of a ribosomal recognition site [Shine-Dalgarno (SD)]. In the simplest form, the SD sequence overlaps with the metabolite-sensitive aptamer domain at its downstream end. Representative examples include the S-adenosylmethionine class II (SAM-II) (10) and the S-adenosylhomocysteine (SAH) riboswitches (11, 12), as well as prequeuosine class I (preQ 1 -I) and II (preQ 1 -II) riboswitches (13,14). The secondary structures of these four short RNA families contain a pseudoknot fold that is central to their gene regulation capacity. Although the SAM-II and preQ 1 -I riboswitches fold into classical pseudoknots (15, 16), the conformations of the SAH (17) and preQ 1 -II counterparts are more complex and include a structural extension that contributes to the pseudoknot architecture (14). Importantly, the impact and evolutionary significance of these "extra" stem-loop elements on the function of the SAH and preQ 1 -II riboswitches remain unclear.PreQ 1 riboswitches interact with the bacterial metabolite 7-aminomethyl-7-deazaguanine (preQ 1 ), a precursor molecule in the biosynthetic pathway of queuosine, a modified base encountered at the wobble position of some transfer RNAs (14). The general biological significance of studying the preQ 1 -II system stems from the fact that this gene-regulatory element is found...

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“…Therefore, studying RNA unfolding/refolding becomes a pertinent issue of RNA biology. To meet such a challenge, the single-molecule approach has been employed to unravel RNA structural polymorphism by studying one molecule at a time [22][23][24][25][26][27] .…”

Section: Introductionmentioning

confidence: 99%

Nanomanipulation of Single RNA Molecules by Optical Tweezers

Stephenson

Wan

Tenenbaum

et al. 2014

JoVE

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A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed. Video LinkThe video component of this article can be found at

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Allosteric Tertiary Interactions Preorganize the c-di-GMP Riboswitch and Accelerate Ligand Binding

Cited by 62 publications

References 55 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

Tuning a riboswitch response through structural extension of a pseudoknot

Nanomanipulation of Single RNA Molecules by Optical Tweezers

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