Phosphate‐Group Recognition by the Aptamer Domain of the Thiamine Pyrophosphate Sensing Riboswitch

Noeske, J.; Richter, Christian; Stirnal, Elke; Schwalbe, Harald; Wöhnert, Jens

doi:10.1002/cbic.200600151

Cited by 27 publications

(19 citation statements)

References 39 publications

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“…S5) for the WT aptamer binding to the substrates thiamine (T) and thiamine monophosphate (TMP). These alternative ligands carry fewer phosphates than TPP and are unable to bind the P4∕5 arm via native, magnesium-mediated contacts (19,(26)(27)(28). Nevertheless, the WT aptamer displayed similar energetic stabilization by either T or TMP, when present at saturating concentrations, as did the A90G aptamer bound to TPP (Fig.…”

Section: Resultsmentioning

confidence: 94%

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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Section: Resultsmentioning

confidence: 94%

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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“…For instance for the thiamine pyrophosphate (TPP)-sensing riboswitch it was shown that Mg 2+ binding is absolutely required for TPP binding (4) and cannot be replaced by monovalent ions or Ca 2+ (5). The X-ray structure of the thiC TPP-sensing riboswitch from Arabidopsis thaliana bound to TPP reveals that one Mg 2+ -ion chelates oxygen atoms in the pyrophosphate moiety of TPP and links them to the RNA whereas there are two bridging Mg 2+ -ions in the X-ray structure of the thiM TPP-sensing riboswitch from Escherichia coli bound to TPP.…”

Section: Introductionmentioning

confidence: 99%

Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain

Noeske

Schwalbe

Wöhnert

2007

Nucleic Acids Research

127

120

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Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg2+-ions. Furthermore, a role for Mg2+-ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNA–adenine complex in solution as studied by high-resolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg2+, but becomes partially pre-organized for ligand binding in the presence of Mg2+. Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg2+ is more pronounced.

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“…[7][8][9] However, to understand how these RNAs function, analysis of the complete TPP riboswitch with and without its ligand is required. However, the ligand-free structure is difficult to refine by NMR spectroscopy or crystallography.…”

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confidence: 99%

RNA Ligands That Distinguish Metabolite‐Induced Conformations in the TPP Riboswitch

et al. 2007

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Riboswitches are conserved noncoding elements, located in untranslated regions of messenger RNAs (mRNAs), which regulate the expression of metabolism genes in biosynthetic pathways in response to metabolite binding. [1,2] They bind to small metabolite molecules, thereby triggering the expression of distinct genes that are in turn involved in biosynthetic pathways of the metabolite. As riboswitches regulate the expression of essential genes in many bacteria, these RNAs might serve as targets for the development of novel antimicrobial drugs. [3][4][5] The thiM riboswitch of E. coli regulates the expression of the metabolic protein hydroxyethylthiazolkinase and is known to inhibit gene expression mainly by the sequestration of the Shine-Dalgarno (SD) sequence induced by specific binding of thiamine pyrophosphate (TPP) to an aptamer domain.[6] However, like other metabolite-dependent riboswitches, thiM riboswitches also contain an expression domain which does not directly interact with TPP but is required for proper riboswitch function by a mechanism in which TPPinduced structural rearrangements are thought to be relayed to this domain, thereby interfering with gene expression.Crystallographic and NMR spectroscopy analyses of the aptamer domain of TPP riboswitches in complex with its ligand TPP provided insight into how riboswitch aptamer domains create specific binding pockets. [7][8][9] However, to understand how these RNAs function, analysis of the complete TPP riboswitch with and without its ligand is required. However, the ligand-free structure is difficult to refine by NMR spectroscopy or crystallography.[10] Therefore, molecular probes that can distinguish between activation states of riboswitches might help to identify sites that are involved in conformational changes and thus, might be useful for gaining insight into the mechanisms of riboswitch function.One class of molecular probes that can be obtained by in vitro selection are RNA aptamers-single stranded nucleic acids that fold into a distinct 3D-structure and are able to bind with high affinity and specificity to a cognate target molecule.[11] Aptamers have been isolated against a variety of targets including small molecules, peptides, proteins, and living cells [12] and they can discriminate between activation states of proteins.[13] However, no RNA aptamer has been reported to date that is able to differentiate between RNA conformations.Herein, we describe the isolation of short RNA molecules that are able to bind to the TPP-free thiM riboswitch of E. coli and are released upon riboswitch-metabolite complex formation. We applied a modified in vitro selection procedure in which the 165-nucleotide (nt) thiM riboswitch was biotinylated at its 5'-end and immobilized on streptavidin magnetic beads (Scheme 1). These beads were incubated with a RNA library comprising a 25-nt random region. To avoid the participation of the constant regions of the RNA library in thiM binding, we sequestered these regions by hybridization to complementary oligodeoxynucleotide...

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Phosphate‐Group Recognition by the Aptamer Domain of the Thiamine Pyrophosphate Sensing Riboswitch

Cited by 27 publications

References 39 publications

Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer

Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer

Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain

RNA Ligands That Distinguish Metabolite‐Induced Conformations in the TPP Riboswitch

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