Metal-Induced Folding of Diels−Alderase Ribozymes Studied by Static and Time-Resolved NMR Spectroscopy

Manoharan, Vijayalaxmi; Fürtig, Boris; Jäschke, Andres; Schwalbe, Harald

doi:10.1021/ja900244x

Cited by 22 publications

(31 citation statements)

References 55 publications

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“…The possibility to disassemble the catalytic pocket by breaking a single hydrogen bond may be important for the multiple-turnover behavior of this catalyst (8,9), and also for the possibility of a ‘back door’ reaction pathway that requires substrate passage through a narrow orifice (29). Such a frequent breakdown and re-formation of the tertiary structure would be consistent with the structural transitions between ‘folded’ and ‘intermediate’ conformations on the 100 ms timescale, which were observed by both single-molecule (12) and NMR spectroscopy (13). …”

Section: Discussionsupporting

confidence: 72%

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Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

Kraut¹,

Bebenroth²,

Nierth³

et al. 2011

Nucleic Acids Research

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Compared to protein enzymes, our knowledge about how RNA accelerates chemical reactions is rather limited. The crystal structures of a ribozyme that catalyzes Diels–Alder reactions suggest a rich tertiary architecture responsible for catalysis. In this study, we systematically probe the relevance of crystallographically observed ground-state interactions for catalytic function using atomic mutagenesis in combination with various analytical techniques. The largest energetic contribution apparently arises from the precise shape complementarity between transition state and catalytic pocket: A single point mutant that folds correctly into the tertiary structure but lacks one H-bond that normally stabilizes the pocket is completely inactive. In the rate-limiting chemical step, the dienophile is furthermore activated by two weak H-bonds that contribute ∼7–8 kJ/mol to transition state stabilization, as indicated by the 25-fold slower reaction rates of deletion mutants. These H-bonds are also responsible for the tight binding of the Diels–Alder product by the ribozyme that causes product inhibition. For high catalytic activity, the ribozyme requires a fine-tuned balance between rigidity and flexibility that is determined by the combined action of one inter-strand H-bond and one magnesium ion. A sharp 360° turn reminiscent of the T-loop motif observed in tRNA is found to be important for catalytic function.

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Section: Discussionsupporting

confidence: 72%

“…Single-molecule fluorescence resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopy have revealed that the ribozyme is highly dynamic in aqueous solution at room temperature (12,13). The concentration of divalent metal ions was established as a major determinant of folding and dynamics (12–14).…”

Section: Introductionmentioning

confidence: 99%

Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

Kraut¹,

Bebenroth²,

Nierth³

et al. 2011

Nucleic Acids Research

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“…4 Therefore, catalysis may not be decoupled from RNA folding. This latter process is hierarchical, first proceeding on the secondary structure level via formation of fairly stable Watson–Crick base pairs.…”

Section: Introductionmentioning

confidence: 99%

Complex RNA Folding Kinetics Revealed by Single-Molecule FRET and Hidden Markov Models

et al. 2014

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We have developed a hidden Markov model and optimization procedure for photon-based single-molecule FRET data, which takes into account the trace-dependent background intensities. This analysis technique reveals an unprecedented amount of detail in the folding kinetics of the Diels–Alderase ribozyme. We find a multitude of extended (low-FRET) and compact (high-FRET) states. Five states were consistently and independently identified in two FRET constructs and at three Mg2+ concentrations. Structures generally tend to become more compact upon addition of Mg2+. Some compact structures are observed to significantly depend on Mg2+ concentration, suggesting a tertiary fold stabilized by Mg2+ ions. One compact structure was observed to be Mg2+-independent, consistent with stabilization by tertiary Watson–Crick base pairing found in the folded Diels–Alderase structure. A hierarchy of time scales was discovered, including dynamics of 10 ms or faster, likely due to tertiary structure fluctuations, and slow dynamics on the seconds time scale, presumably associated with significant changes in secondary structure. The folding pathways proceed through a series of intermediate secondary structures. There exist both compact pathways and more complex ones, which display tertiary unfolding, then secondary refolding, and, subsequently, again tertiary refolding.

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“…In addition, the nucleobase carbon (C2, C5, C6, and C8) and proton (H2, H5, H6, and H8) chemical shifts are increasingly being used in defining the 3D structures of RNA and DNA (Wijmenga et al 1997; Cromsigt et al 2001; Xu and Case 2001; Barton et al 2013; Frank et al 2013; Frank et al 2013; Sahakyan and Vendruscolo 2013; Werf et al 2013; Sripakdeevong et al 2014). Having the ability to acquire these chemical shift data rapidly would be important for applications ranging from nucleic acid targeted ligand screening to time-resolved NMR studies of biochemical processes such as catalysis (Buck et al 2009) and folding (Wenter et al 2005; Buck et al 2007; Manoharan et al 2009; Lee et al 2010; Lieblein et al 2012; Li et al 2014). In addition, L-optimization for aromatic protons has been successfully implemented to measure residual dipolar couplings for nucleic acids (Ying et al 2011).…”

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confidence: 99%

Development and application of aromatic [13C, 1H] SOFAST-HMQC NMR experiment for nucleic acids

et al. 2014

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Higher sensitivity of NMR spectrometers and novel isotopic labeling schemes have ushered the development of rapid data acquisition methodologies, improving the time resolution with which NMR data can be acquired. For nucleic acids, longitudinal relaxation optimization in conjunction with Ernst angle excitation (SOFAST-HMQC) for imino protons, in addition to rendering rapid pulsing, has been demonstrated to yield significant improvements in sensitivity per unit time. Extending such methodology to other spins offers a viable prospect to measure additional chemical shifts, thereby broadening their utilization for various applications. Here, we introduce the 2D [13C,1H] aromatic SOFAST-HMQC that results in overall sensitivity gain of 1.4–1.7 fold relative to the conventional HMQC and can also be extended to yield long-range heteronuclear chemical shifts such as the adenine imino nitrogens N1, N3, N7 and N9. The applications of these experiments range from monitoring real-time biochemical processes, drug/ligand screening, and to collecting data at very low sample concentration and/or in cases where isotopic enrichment cannot be achieved.

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Metal-Induced Folding of Diels−Alderase Ribozymes Studied by Static and Time-Resolved NMR Spectroscopy

Cited by 22 publications

References 55 publications

Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

Three critical hydrogen bonds determine the catalytic activity of the Diels–Alderase ribozyme

Complex RNA Folding Kinetics Revealed by Single-Molecule FRET and Hidden Markov Models

Development and application of aromatic [13C, 1H] SOFAST-HMQC NMR experiment for nucleic acids

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