A General Approach for the Identification of Site‐Specific RNA Binders by <sup>19</sup>F NMR Spectroscopy: Proof of Concept

Kreutz, Christoph; Kählig, Hanspeter; Konrat, Robert; Micura, Ronald

doi:10.1002/anie.200504174

Cited by 68 publications

(62 citation statements)

References 38 publications

(46 reference statements)

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“…Initial attempts to segmentally label the expression platform with 15 N∕ 13 C nucleotides by a combined approach using enzymatic transcription (T7 RNA polymerase) and enzymatic ligation (21,22) to the chemically synthesized aptamer domain failed. Alternatively, we decided to employ a 19 F NMR spectroscopic approach (23)(24)(25)(26) for the purpose to analyze how the equilibrium structures respond to preQ 1 recognition. Because uridine at position 37 is ideally positioned in the sense that this pyrimidine is base-paired within one of the two proposed secondary structures while being unpaired in the alternative, we anticipated a straightforward assignment and quantification of the equilibrium structures using the noninvasive 5-fluoro pyrimidine label (Fig.…”

Section: Resultsmentioning

confidence: 99%

Folding of a transcriptionally acting PreQ ₁ riboswitch

Rieder

Kreutz

Micura

2010

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

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104

133

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7-Aminomethyl-7-deazaguanine (preQ 1 ) sensitive mRNA domains belong to the smallest riboswitches known to date. Although recent efforts have revealed the three-dimensional architecture of the ligand-aptamer complex less is known about the molecular details of the ligand-induced response mechanism that modulates gene expression. We present an in vitro investigation on the ligand-induced folding process of the preQ 1 responsive RNA element from Fusobacterium nucleatum using biophysical methods, including fluorescence and NMR spectroscopy of site-specifically labeled riboswitch variants. We provide evidence that the full-length riboswitch domain adopts two different coexisting stem-loop structures in the expression platform. Upon addition of preQ 1 , the equilibrium of the competing hairpins is significantly shifted. This system therefore, represents a finely tunable antiterminator/terminator interplay that impacts the in vivo cellular response mechanism. A model is presented how a riboswitch that provides no obvious overlap between aptamer and terminator stem-loop solves this communication problem by involving bistable sequence determinants.RNA | chemical synthesis | labeling | bistable structures | riboswitches N oncoding regions of mRNA that bind metabolites with high selectivity and specificity function as so-called riboswitches (1-3). They represent gene regulation systems that are widespread among bacteria and importantly, they do not rely on the assistance of proteins (4). Riboswitches consist of a metabolite-sensitive aptamer and an adjoining expression platform (5) Although impressive progress has been made in revealing three-dimensional structures of metabolite-bound aptamer complexes of various riboswitch classes (6), less is known about how binding of the metabolite to the aptamer is communicated into a structural change of the expression platform, which in turn signals "on" or "off" for gene expression. The simplified picture of bacterial transcription control is that upon metabolite binding either a mRNA terminator structure is formed, which causes the RNA polymerase to stop synthesis (off regulation) or an existing terminator is disrupted, which enables the polymerase to continue synthesis with the mRNA template (on regulation) (5, 7). In the case of translational control, accessibility versus sequestration of the Shine-Dalgarno sequence upon metabolite binding is the essence of the response mechanism for on versus off regulation, corresponding to hindrance or enabling of bacterial ribosome translation initiation (5, 7-9).The preQ 1 class I riboswitch is the smallest riboswitch known to date (10). Its aptamer comprises a stretch of only 34 nucleotides in the 5′ untranslated leader region of the respective messenger RNA and specifically recognizes 7-aminomethyl-7-deazaguanine (preQ 1 ), which is an intermediate in queuosine biosynthesis (11). The minimal sequence and structure consensus refer to a hairpin comprising a five base-pair stem (P1) and a loop of eleven to thirteen nucleotides (L1) together...

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Section: Resultsmentioning

confidence: 99%

Folding of a transcriptionally acting PreQ ₁ riboswitch

Rieder

Kreutz

Micura

2010

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

Self Cite

104

133

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“…Kreutz et al [104] applied 19 F NMR spectroscopy to identify site-specific ligand-RNA binding. The approach relied on site-specific labelling of RNA with 2'-deoxy-2'fluoro(2'-F) nucleosides, thereby replacing the 2'-hydroxy group with a fluorine atom.…”

Section: Ligand-dna Ligand-rna and Ligand Membrane Interactionsmentioning

confidence: 99%

NMR Spectroscopy for Studying Interactions of Bioactive Molecules

Novak¹,

Jednačak²

2012

Physico-Chemical Methods in Drug Discovery and Development

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“…19 F-labeled nucleotides are often introduced by chemical synthesis which provides a single introduction of the label at a defined position within the RNA of interest. 19 F-nuclei are very sensitive to environmental changes such as differences in the secondary structure or ligand binding (Kreutz et al 2005(Kreutz et al , 2006. Together with the rather small size of the label it represents a very useful tool for the analysis of bistable RNAs, weak complexes, or large ligand binding RNAs (Graber et al 2008;Kiviniemi and Virta 2011).…”

Section: Introductionmentioning

confidence: 99%

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

et al. 2015

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In comparison to proteins and protein complexes, the size of RNA amenable to NMR studies is limited despite the development of new isotopic labeling strategies including deuteration and ligation of differentially labeled RNAs. Due to the restricted chemical shift dispersion in only four different nucleotides spectral resolution remains limited in larger RNAs. Labeling RNAs with the NMR-active nucleus 19

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A General Approach for the Identification of Site‐Specific RNA Binders by ¹⁹F NMR Spectroscopy: Proof of Concept

Cited by 68 publications

References 38 publications

Folding of a transcriptionally acting PreQ ₁ riboswitch

Folding of a transcriptionally acting PreQ ₁ riboswitch

NMR Spectroscopy for Studying Interactions of Bioactive Molecules

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

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A General Approach for the Identification of Site‐Specific RNA Binders by 19F NMR Spectroscopy: Proof of Concept

Cited by 68 publications

References 38 publications

Folding of a transcriptionally acting PreQ 1 riboswitch

Folding of a transcriptionally acting PreQ 1 riboswitch

NMR Spectroscopy for Studying Interactions of Bioactive Molecules

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

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A General Approach for the Identification of Site‐Specific RNA Binders by ¹⁹F NMR Spectroscopy: Proof of Concept

Folding of a transcriptionally acting PreQ ₁ riboswitch

Folding of a transcriptionally acting PreQ ₁ riboswitch