Single-Molecule Studies of the Lysine Riboswitch Reveal Effector-Dependent Conformational Dynamics of the Aptamer Domain

Fiegland, Larry R.; Garst, Andrew D.; Batey, Robert T.; Nesbitt, David J.

doi:10.1021/bi3007753

Cited by 45 publications

(78 citation statements)

References 63 publications

(166 reference statements)

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“…Typically, a riboswitch aptamer domain is site-specifically labeled with both donor and acceptor fluorophores for FRET, then attached to a glass (or quartz) surface via linker chemistry (Figure 2) [51]. To date, lysine [52], SAM class I [53], SAM class II [54], guanine [55], adenine [56–58], TPP [59], c-di-GMP class I [60], and preQ 1 class I & II riboswitches [61, 62] have been explored using smFRET methodologies (section 4 ).…”

Section: Single-molecule Methodsmentioning

confidence: 99%

Single-molecule studies of riboswitch folding

Savinov

Perez

Block

2014

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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The folding dynamics of riboswitches are central to their ability to modulate gene expression in response to environmental cues. In most cases, a structural competition between the formation of a ligand-binding aptamer and an expression platform (or some other competing off-state) determines the regulatory outcome. Here, we review single-molecule studies of riboswitch folding and function, predominantly carried out using single-molecule FRET or optical trapping approaches. Recent results have supplied new insights into riboswitch folding energy landscapes, the mechanisms of ligand binding, the roles played by divalent ions, the applicability of hierarchical folding models, and kinetic vs. thermodynamic control schemes. We anticipate that future work, based on improved data sets and potentially combining multiple experimental techniques, will enable the development of more complete models for complex RNA folding processes.

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Section: Single-molecule Methodsmentioning

confidence: 99%

Single-molecule studies of riboswitch folding

Savinov

Perez

Block

2014

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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“…For the SAM-I riboswitch, a dynamic three-state exchange process was uncovered where the ligand-free state exhibited unfolding dynamics in the milliseconds regime and the most compacted fold was frozen in the presence of ligand (43). For the lysine riboswitch, smFRET was used to measure the opening/closing rate constants for the aptamer domain as well as the lysine dissociation constant (9). Although these studies have provided an important foundation for understanding the relationship between riboswitch dynamics, ligand binding and gene expression regulation, continued, indepth investigations along these lines of pursuit are greatly needed.…”

Section: Large Structurally Complex Riboswitches With Pseudoknots Andmentioning

confidence: 99%

“…In this form of regulation, the target mRNA typically undergoes a structural change in response to metabolite binding (5)(6)(7)(8)(9). These mRNA elements have thus been termed "riboswitches" and generally include both a metabolite-sensitive aptamer subdomain and an expression platform.…”

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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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“…66,78 Fiegland and colleagues showed that ligand binding promotes a population shift into a previously unoccupied high-FRET state that corresponds to the close association of the bottom stem-loops, consistent with the 'ordering' definition of IF. 79 In addition, as the concentration of lysine increases, the folding rate also increases, 79 while the unfolding rate is insensitive to the ligand concentration. This is the definition of IF according to the kinetic/energetic picture.…”

Section: Insights Into Aptamer-ligand Interactions From Single-molecumentioning

confidence: 99%

“…The famous picture of the folding energy landscape is, after all, highdimensional, and dozens of environmental factors, including Mg 2+ and the ligand, influence it. While some riboswitches like lysC have a well-defined mechanism across a wide range of environments, 79 bsu can bind its ligand at different stages of folding, 84 changing its path to the native state dramatically, while env8 86 and the adenine Please do not adjust margins Please do not adjust margins …”

Section: +mentioning

confidence: 99%

An integrated perspective on RNA aptamer ligand-recognition models: clearing muddy waters

McCluskey

Penedo

2017

Phys. Chem. Chem. Phys.

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Riboswitches are short RNA motifs that sensitively and selectively bind cognate ligands to modulate gene expression. Like protein receptor-ligand pairs, their binding dynamics are traditionally categorized as following one of two paradigmatic mechanisms: conformational selection and induced fit. In conformational selection, ligand binding stabilizes a particular state already present in the receptor's dynamic ensemble. In induced fit, ligand-receptor interactions enable the system to overcome the energetic barrier into a previously inaccessible state. In this article, we question whether a polarized division of RNA binding mechanisms truly meets the conceptual needs of the field. We will review the history behind this classification of RNA-ligand interactions, and the way induced fit in particular has been rehabilitated by single-molecule studies of RNA aptamers. We will highlight several recent results from single-molecule experimental studies of riboswitches that reveal gaps or even contradictions between common definitions of the two terms, and we will conclude by proposing a more robust framework that considers the range of RNA behaviors unveiled in recent years as a reality to be described, rather than an increasingly unwieldy set of exceptions to the traditional models.

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Single-Molecule Studies of the Lysine Riboswitch Reveal Effector-Dependent Conformational Dynamics of the Aptamer Domain

Cited by 45 publications

References 63 publications

Single-molecule studies of riboswitch folding

Single-molecule studies of riboswitch folding

Tuning a riboswitch response through structural extension of a pseudoknot

An integrated perspective on RNA aptamer ligand-recognition models: clearing muddy waters

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