Janina Buck

Goethe-Universität Frankfurt am Main, Frankfurt am Main, Hesse, Germany

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Publications (22)120.49 Total impact

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    ABSTRACT: Riboswitches are elements in the 5'-untranslated region of mRNAs that regulate gene expression by directly interacting with metabolites related to their own gene products. A remarkable feature of this gene regulation mechanism is the high specificity of riboswitches for their cognate ligands. In this study, we used a combination of static and time-resolved NMR-spectroscopic methods to investigate the mechanisms for ligand specificity in purine riboswitches. We investigate the xpt-aptamer domain from a guanine-responsive riboswitch and the mfl-aptamer domain from a 2'-deoxyguanosine-responsive riboswitch. The xpt-aptamer binds the purine nucleobases guanine/hypoxanthine with high affinity, but, unexpectedly, also the nucleoside 2'-deoxyguanosine. On the other hand, the mfl-aptamer is highly specific for its cognate ligand 2'-deoxyguanosine, and does not bind purine ligands. We addressed the question of aptamer`s ligand specificity by real-time NMR spectroscopy. Our studies of ligand binding and subsequently induced aptamer folding revealed that the xpt-aptamer discriminates against non-cognate ligands by enhanced life-times of the cognate complex compared with non-cognate complexes, whereas the mfl-aptamer rejects non-cognate ligands at the level of ligand association, employing a kinetic proofreading mechanism.
    RNA biology 05/2012; 9(5):672-80. · 5.56 Impact Factor
  • Angewandte Chemie 01/2012; 124(1).
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    ABSTRACT: Catalytic RNA motifs (ribozymes) are involved in various cellular processes. Although functional cleavage of the RNA phosphodiester backbone for self-cleaving ribozymes strongly differs with respect to sequence specificity, the structural context, and the underlying mechanism, these ribozyme motifs constitute evolved RNA molecules that carry out identical chemical functionality. Therefore, they represent ideal systems for detailed studies of the underlying structure-function relationship, illustrating the diversity of RNA's functional role in biology. Nuclear magnetic resonance (NMR) spectroscopic methods in solution allow investigation of structure and dynamics of functional RNA motifs at atomic resolution. In addition, characterization of RNA conformational transitions initiated either through addition of specific cofactors, as e.g. ions or small molecules, or by photo-chemical triggering of essential RNA functional groups provides insights into the reaction mechanism. Here, we discuss applications of static and time-resolved NMR spectroscopy connected with the design of suitable NMR probes that have been applied to characterize global and local RNA functional dynamics together with cleavage-induced conformational transitions of two RNA ribozyme motifs: a minimal hammerhead ribozyme and an adenine-dependent hairpin ribozyme.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 848:185-99. · 1.29 Impact Factor
  • Biophysical Journal 01/2012; 102(3):277-. · 3.67 Impact Factor
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    ABSTRACT: Get the zipper right: Time-resolved NMR spectroscopy measurements reveal kinetic partitioning for pH-induced DNA i-motif formation. The data characterize the folding pathway of i-motifs and provide a biophysical based description of pH-dependent folding processes as utilized in live-cell pH-sensors.
    Angewandte Chemie International Edition 11/2011; 51(1):250-3. · 11.34 Impact Factor
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    ABSTRACT: Among the three major classes of biomacromolecules (DNA, RNA, and proteins) RNA's pronounced dynamics are the most explicitly linked to its wide variety of functions, which include catalysis and the regulation of transcription, translation, and splicing. These functions are mediated by a range of RNA biomachinery, including such varied examples as macromolecular noncoding RNAs, microRNAs, small interfering RNAs, riboswitch RNAs, and RNA thermometers. In each case, the functional dynamics of an interconversion is characterized by an associated rate constant. In this Account, we provide an introduction to NMR spectroscopic characterization of the landscape of RNA dynamics. We introduce strategies for measuring NMR parameters at various time scales as well as the underlying models for describing the corresponding rate constants. RNA exhibits significant dynamic motion, which can be modulated by (i) intermolecular interactions, including specific and nonspecific binding of ions (such as Mg(2+) and tertiary amines), (ii) metabolites in riboswitches or RNA aptamers, and (iii) macromolecular interactions within ribonucleic protein particles, including the ribosome and the spliceosome. Our understanding of the nature of these dynamic changes in RNA targets is now being incorporated into RNA-specific approaches in the design of RNA inhibitors. Interactions of RNA with proteins, other RNAs, or small molecules often occur through binding mechanisms that follow an induced fit mechanism or a conformational selection mechanism, in which one of several populated RNA conformations is selected through ligand binding. The extent of functional dynamics, including the kinetic formation of a specific RNA tertiary fold, is dependent on the messenger RNA (mRNA) chain length. Thus, during de novo synthesis of mRNA, both in prokaryotes and eukaryotes, nascent mRNA of various lengths will adopt different secondary and tertiary structures. The speed of transcription has a critical influence on the functional dynamics of the RNA being synthesized. In addition to modulating the local dynamics of a conformational RNA ensemble, a given RNA sequence may adopt more than one global, three-dimensional structure. RNA modification is one way to select among these alternative structures, which are often characterized by nearly equal stability, but with high energy barriers for conformational interconversion. The refolding of different secondary and tertiary structures has been found to be a major regulatory mechanism for transcription and translation. These conformational transitions can be characterized with NMR spectroscopy, for any given RNA sequence, in response to external stimuli.
    Accounts of Chemical Research 09/2011; 44(12):1292-301. · 20.83 Impact Factor
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    ABSTRACT: Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop-loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gsw(loop)) in the absence of Mg(2+). However, if Mg(2+) is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop-loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gsw(loop) is tunable through variation of the Mg(2+) concentration. We quantitatively describe the influence of distinct Mg(2+) concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution.
    Nucleic Acids Research 09/2011; 39(22):9768-78. · 8.81 Impact Factor
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    ABSTRACT: The mfl-riboswitch regulates expression of ribonucleotide reductase subunit in Mesoplasma florum by binding to 2'-deoxyguanosine and thereby promoting transcription termination. We characterized the structure of the ligand-bound aptamer domain by NMR spectroscopy and compared the mfl-aptamer to the aptamer domain of the closely related purine-sensing riboswitches. We show that the mfl-aptamer accommodates the extra 2'-deoxyribose unit of the ligand by forming a more relaxed binding pocket than these found in the purine-sensing riboswitches. Tertiary structures of the xpt-aptamer bound to guanine and of the mfl-aptamer bound to 2'-deoxyguanosine exhibit very similar features, although the sequence of the mfl-aptamer contains several alterations compared to the purine-aptamer consensus sequence. These alterations include the truncation of a hairpin loop which is crucial for complex formation in all purine-sensing riboswitches characterized to date. We further defined structural features and ligand binding requirements of the free mfl-aptamer and found that the presence of Mg(2+) is not essential for complex formation, but facilitates ligand binding by promoting pre-organization of key structural motifs in the free aptamer.
    Nucleic Acids Research 05/2011; 39(15):6802-12. · 8.81 Impact Factor
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    ABSTRACT: NMR-spektroskopische Untersuchungen an regulatorischen RNAs geben Aufschluss über den mechanistischen Zusammenhang von Faltungswegen, dreidimensionaler Struktur, Dynamik und Funktion in Genregulationsprozessen. NMR spectroscopic measurements reveal how folding, structure, molecular dynamics, and function in regulatory RNAs are interconnected.
    BioSpektrum 03/2011; 17(2):174-177.
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    ABSTRACT: We present here a set of (13)C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose (13)C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4' nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1',H1' ribose signals. The experiments were applied to two RNA hairpin structures. The current set of (13)C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, (13)C-direct detected NMR methods constitute useful complements to the conventional (1)H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.
    Journal of Biomolecular NMR 08/2010; 47(4):259-69. · 2.85 Impact Factor
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    ABSTRACT: Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated pre-formation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg(2+) for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gsw(loop)) of the guanine-sensing riboswitch and their Mg(2+)-induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gsw(loop) or the increase in temperature for both Gsw and Gsw(loop) involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg(2+). We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding.
    Nucleic Acids Research 03/2010; 38(12):4143-53. · 8.81 Impact Factor
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    ABSTRACT: In-cell NMR spectroscopy of proteins in different cellular environments is a well-established technique that, however, has not been applied to nucleic acids so far. Here, we show that isotopically labeled DNA and RNA can be observed inside the eukaryotic environment of Xenopus laevis oocytes by in-cell NMR spectroscopy. One limiting factor for the observation of nucleic acids in Xenopus oocytes is their reduced stability. We demonstrate that chemical modification of DNA and RNA can protect them from degradation and can significantly enhance their lifetime. Finally, we show that the imino region of the NMR spectrum is devoid of any oocyte background signals enabling the detection even of isotopically nonlabeled molecules.
    Journal of the American Chemical Society 10/2009; 131(43):15761-8. · 10.68 Impact Factor
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    ABSTRACT: Time-resolved NMR spectroscopy was applied to study ribozyme-mediated RNA catalysis in a mutant of the hairpin ribozyme, the adenine-dependent hairpin ribozyme (ADHR; M. Meli, et al. J. Biol. Chem. 2003, 278, 9835-9842) with atomic resolution. The mutant ADHR was designed to investigate the role of cofactors in RNA catalytic mechanisms in order to understand cellular processes that could have been present in the archaic "RNA world" and of their evolution towards functional RNAs in modern cellular processes, as for example, found in the glmS ribozyme. Conformational changes due to RNA cleavage were analyzed following spectral changes of the NMR imino proton resonances that could be assigned both for the pre- and postcleaved conformation for this 80-nucleotide long RNA. (31)P NMR spectroscopic studies allowed us to confirm the formation of a cyclic phosphodiester as a result of the cleavage process. For ADHR, both metal ions and the cofactor adenine are essential for self-cleaving activity. The interaction of the ribozyme with the cofactor adenine is found to be transient and too weak to significantly change the RNA structure or to modulate the spectroscopic characteristics of the cofactor. ADHR therefore represents a ribozyme in which high activation barriers have to be overcome to populate cleavage-competent states that exhibit short life times. We show that conformational dynamics, but not the chemistry, constitute the rate-limiting step in catalysis of the adenine-dependent hairpin ribozyme.
    ChemBioChem 08/2009; 10(12):2100-10. · 3.74 Impact Factor
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    ABSTRACT: A detailed understanding of cellular mechanisms requires knowledge of structure and dynamics of the involved biomacromolecules at atomic resolution. NMR spectroscopy uniquely allows determination of static and dynamic processes at atomic level, including structured states often represented by a single state as well as by unstructured conformational ensembles. While a high-resolution description of structured states may also be obtained by other techniques, the characterization of structural transitions occurring during biomolecular folding is only feasible exploiting NMR spectroscopic methods. The NMR methodical strategy includes the fast initiation of a folding reaction in situ and the possibility to detect the induced process with sufficient time resolution on the respective NMR time scale. In the case of ligand-induced structural transitions of RNA, the initiation of the folding reaction can be achieved by laser-triggered deprotection of a photolabile caged ligand whose release induces folding of a riboswitch RNA. The strategy discussed here is general and can also be transferred to other biological processes, where at least one key reagent or substrate, e.g., ions, ligands, pH, or one specific conformational state, can be photochemically caged. The rates of reversible and irreversible reactions or structural transitions that can be covered by real-time NMR methods range from milliseconds up to hours.In this chapter, we discuss the application of a time-resolved NMR strategy to resolve the ligand-induced folding of the guanine-sensing riboswitch aptamer domain of the B. subtilis xpt-pbuX operon.
    Methods in molecular biology (Clifton, N.J.) 02/2009; 540:161-71. · 1.29 Impact Factor
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    ABSTRACT: Structural transitions of RNA between alternate conformations with similar stabilities are associated with important aspects of cellular function. Few techniques presently exist that are capable of monitoring such transitions and thereby provide insight into RNA dynamics and function at atomic resolution. Riboswitches are found in the 5'-UTR of mRNA and control gene expression through structural transitions after ligand recognition. A time-resolved NMR strategy was established in conjunction with laser-triggered release of the ligand from a photocaged derivative in situ to monitor the hypoxanthine-induced folding of the guanine-sensing riboswitch aptamer domain of the Bacillus subtilis xpt-pbuX operon at atomic resolution. Combining selective isotope labeling of the RNA with NMR filter techniques resulted in significant spectral resolution and allowed kinetic analysis of the buildup rates for individual nucleotides in real time. Three distinct kinetic steps associated with the ligand-induced folding were delineated. After initial complex encounter the ligand-binding pocket is formed and results in subsequent stabilization of a remote long-range loop-loop interaction. Incorporation of NMR data into experimentally restrained molecular dynamics simulations provided insight into the RNA structural ensembles involved during the conformational transition.
    Proceedings of the National Academy of Sciences 11/2007; 104(40):15699-704. · 9.81 Impact Factor
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    ABSTRACT: RNA switches (riboswitches) have important functions in gene regulation. They comprise an aptamer domain, which is responsible for ligand binding, and an expression platform that transmits the ligand-binding state of the aptamer domain through a conformational change. Riboswitches can regulate gene expression either at the level of transcription or translation, and it has been proposed that riboswitch mechanisms are even used to regulate the processing of mRNA. This Minireview summarizes the current understanding of the structures and mode of action of RNA switches, with particular focus on secondary and tertiary interactions, which stabilize the global RNA structure and thus determine the function of the aptamer domain.
    Angewandte Chemie International Edition 02/2007; 46(8):1212-9. · 11.34 Impact Factor
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    ABSTRACT: Riboswitches are highly structured elements in the 5'-untranslated regions (5'-UTRs) of messenger RNA that control gene expression by specifically binding to small metabolite molecules. They consist of an aptamer domain responsible for ligand binding and an expression platform. Ligand binding in the aptamer domain leads to conformational changes in the expression platform that result in transcription termination or abolish ribosome binding. The guanine riboswitch binds with high-specificity to guanine and hypoxanthine and is among the smallest riboswitches described so far. The X-ray-structure of its aptamer domain in complex with guanine/hypoxanthine reveals an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base pairing interactions. We analyzed the conformational transitions of the aptamer domain induced by binding of hypoxanthine using high-resolution NMR-spectroscopy in solution. We found that the long-range base pairing interactions are already present in the free RNA and preorganize its global fold. The ligand binding core region is lacking hydrogen bonding interactions and therefore likely to be unstructured in the absence of ligand. Mg2+-ions are not essential for ligand binding and do not change the structure of the RNA-ligand complex but stabilize the structure at elevated temperatures. We identified a mutant RNA where the long-range base pairing interactions are disrupted in the free form of the RNA but form upon ligand binding in an Mg2+-dependent fashion. The tertiary interaction motif is stable outside the riboswitch context.
    Nucleic Acids Research 02/2007; 35(2):572-83. · 8.81 Impact Factor
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    ABSTRACT: RNA-Schalter erfüllen wichtige Funktionen in der Genregulation. Sie bestehen aus einer Aptamerdomäne, die eine spezifische und hochaffine Bindung mit einem Liganden eingeht, und einer Expressionsplattform, die durch eine allosterische Umorientierung auf den Ligandbindungszustand der Aptamerdomäne reagiert. Die Genexpression kann sowohl während der Transkription wie auch während der Translation reguliert werden, außerdem wurde postuliert, dass RNA-Schalter die Prozessierung von mRNA steuern. Dieser Kurzaufsatz fasst den Kenntnisstand über die Strukturen und Regulationsmechanismen von RNA-Schaltern zusammen. Besonderes Augenmerk gilt sekundären und tertiären Wechselwirkungen, die die Faltung der globulären RNA-Struktur stabilisieren und so die Funktion der Aptamerdomänen bestimmen.
    Angewandte Chemie 01/2007; 119(8):1232 - 1240.
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    ABSTRACT: The application of real-time NMR experiments to the study of RNA folding, as reviewed in this article, is relatively new. For many RNA folding events, current investigations suggest that the time scales are in the second to minute regime. In addition, the initial investigations suggest that different folding rates are observed for one structural transition may be due to the hierarchical folding units of RNA. Many of the experiments developed in the field of NMR of protein folding cannot directly be transferred to RNA: hydrogen exchange experiments outside the spectrometer cannot be applied since the intrinsic exchange rates are too fast in RNA, relaxation dispersion experiments on the other require faster structural transitions than those observed in RNA. On the other hand, information derived from time-resolved NMR experiments, namely the acquisition of native chemical shifts, can be readily interpreted in light of formation of a single long-range hydrogen bonding interaction. Together with mutational data that can readily be obtained for RNA and new ligation technologies that enhance site resolution even further, time-resolved NMR may become a powerful tool to decipher RNA folding. Such understanding will be of importance to understand the functions of coding and non-coding RNAs in cells.
    Biopolymers 01/2007; 86(5-6):360-83. · 2.88 Impact Factor
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    ABSTRACT: Riboswitches are highly structured mRNA elements that regulate gene expression upon specific binding of small metabolite molecules. The purine-binding riboswitches bind different purine ligands by forming both canonical Watson—Crick and non-canonical intermolecular base pairs, involving a variety of hydrogen bonds between the riboswitch aptamer domain and the purine ligand. Here, we summarize work on the ligand binding modes of both purine-binding aptamer domains, their con-formational characteristics in the free and ligand-bound forms, and their ligand-induced folding. The adenine- and guanine-binding riboswitch aptamer domains display different conformations in their free forms, despite nearly identical nucleotide loop sequences that form a loop—loop interaction in the ligand-bound forms. Interestingly, the stability of helix II is crucial for the formation of the loop—loop interaction in the free form. A more stable helix II in the guanine riboswitch leads to a preformed loop—loop interaction in its free form. In contrast, a less stable helix II in the adenine riboswitch results in a lack of this loop—loop interaction in the absence of ligand and divalent cations.
    01/1970: pages 229-247;