The bulge region of HIV-1 TAR RNA binds metal ions in solution

Olejniczak, Mikołaj; Gdaniec, Zofia; Fischer, Artur; Grabarkiewicz, Tomasz; Bielecki, Lukasz; Adamiak, Ryszard W.

doi:10.1093/nar/gkf541

Cited by 60 publications

(51 citation statements)

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“…Monotonous variations in 1 H and 13 C chemical shifts were observed (Figure 1(b) and (c)), indicating rapid exchange (10 5 -10 3 s −1 ) between free and Mg 2+ "bound" TAR conformations. In agreement with previous 19 F NMR studies, 32 the largest chemical shift perturbations (>0.1 ppm in 1 H) are observed in and around bulge residues (Figure 1(a), in yellow), while negligible shifts are observed for the UUCG tetraloop. Although it is not possible to distinguish contributions to chemical shift perturbations arising from conformational change and Mg 2+ localization, these results indicate that Mg 2+ primarily binds residues in and around the bulge, consistent with the previously reported X-ray structure of TAR bound to Ca 2+.…”

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confidence: 92%

“…31 More recently, experiments employing 19 F chemical shift perturbations in combination with Pb 2+ -induced RNA cleavage have more directly established that the bulge region of TAR binds , Mg 2+ , and Ca 2+ with increasing specificity in solution. 32 Here, we have employed RDC-NMR to investigate the effects of Mg 2+ on both TAR average conformation and dynamics under solution conditions. Binding of Mg 2+ to TAR was initially examined by recording 2D 13 C-1 H correlation spectra of TAR in the presence of increasing Mg 2+ concentrations.…”

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confidence: 99%

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Mg2+-induced Variations in the Conformation and Dynamics of HIV-1 TAR RNA Probed Using NMR Residual Dipolar Couplings

Al‐Hashimi¹,

Pitt²,

Majumdar³

et al. 2003

Journal of Molecular Biology

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The effects of divalent Mg 2+ on the conformation and dynamics of the stem-loop transactivation response element (TAR) RNA from HIV-1 have been characterized using NMR residual dipolar couplings (RDCs). Order matrix analysis of one bond 13 C-1 H RDCs measured in TAR at [Mg 2+ ]:[TAR] stoichiometric ratios of ~3:1 (TAR(3.0 Mg)) and ~4.5:1 (TAR(4.5 Mg)) revealed that Mg 2+ reduces the average inter-helical angle from 47(±5)° in TAR(free) to 5(±7)° in TAR(4.5 Mg). In contrast to the TAR(free) state, the generalized degree of order for the two stems in TAR(4.5 Mg) is found to be identical within experimental uncertainty, indicating that binding of Mg 2+ leads to an arrest of inter-helical motions in TAR(free). Results demonstrate that RDC-NMR methodology can provide new insight into the effects of Mg 2+ on both the conformation and dynamics of RNA. Keywordstransactivation response element; RNA folding; collective motions; divalent metal ions; nucleic acids By neutralizing backbone electrostatic repulsions, divalent ions such as Mg 2+ can profoundly affect RNA average conformation and plasticity, and thus have important consequences on folding, 1-4 catalysis, 5,6 and recognition. 7 Understanding how divalent ions affect RNA conformation and function at a molecular level requires not only the atomic characterization of typically folded divalent ion bound conformations, but also what is more often an ensemble of partially unfolded divalent ion free conformations. 1-4 However, elucidating high-resolution structures of dynamically inter-converting conformational substates poses significant challenges to techniques such as X-ray crystallography and NMR spectroscopy, which remain by and large, limited in applicability to "static" average structure determination of well folded conformations. While NMR relaxation measurements By providing long-range angular sensitivity to both structure and dynamic fluctuations over a wide range of timescales (

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supporting

confidence: 92%

mentioning

confidence: 99%

Mg2+-induced Variations in the Conformation and Dynamics of HIV-1 TAR RNA Probed Using NMR Residual Dipolar Couplings

Al‐Hashimi¹,

Pitt²,

Majumdar³

et al. 2003

Journal of Molecular Biology

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“…We then exploited different chemical shifts of 5-19 F-incorporated pyrimidine nucleotide, in either a single-or doublestranded region of RNA, which has previously been used to probe the secondary structure of RNA (Horowitz et al 1977;Marshall and Smith 1977;Gollnick et al 1986;Chu et al 1992;Kanyo et al 1996;Sahasrabudhe and Gmeiner 1997;Arnold and Fisher 2000;Hammann et al 2001;Olejniczak et al 2002;Hennig et al 2007;Puffer et al 2009) to demonstrate the presence of an alternative conformation of U2-U6 snRNA complex conclusively. The advantage of observing the 19 F nucleus by NMR is the broader range and the greater sensitivity of fluorine chemical shifts in response to the local environment as compared to those of hydrogen because the fluorine nucleus is surrounded by nine electrons vs. a single electron in hydrogen.…”

Section: Discussionmentioning

confidence: 99%

Conformational heterogeneity of the protein-free human spliceosomal U2-U6 snRNA complex

Zhao

Bachu

Popovic

et al. 2013

RNA

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The complex formed between the U2 and U6 small nuclear (sn)RNA molecules of the eukaryotic spliceosome plays a critical role in the catalysis of precursor mRNA splicing. Here, we have used enzymatic structure probing, 19 F NMR, and analytical ultracentrifugation techniques to characterize the fold of a protein-free biophysically tractable paired construct representing the human U2-U6 snRNA complex. Results from enzymatic probing and 19 F NMR for the complex in the absence of Mg 2+ are consistent with formation of a four-helix junction structure as a predominant conformation. However,19 F NMR data also identify a lesser fraction (up to 14% at 25°C) of a three-helix conformation. Based upon this distribution, the calculated ΔG for interconversion to the four-helix structure from the three-helix structure is approximately −4.6 kJ/mol. In the presence of 5 mM Mg 2+ , the fraction of the three-helix conformation increased to ∼17% and the Stokes radius, measured by analytical ultracentrifugation, decreased by 2%, suggesting a slight shift to an alternative conformation. NMR measurements demonstrated that addition of an intron fragment to the U2-U6 snRNA complex results in displacement of U6 snRNA from the region of Helix III immediately 5 ′ of the ACAGAGA sequence of U6 snRNA, which may facilitate binding of the segment of the intron adjacent to the 5 ′ splice site to the ACAGAGA sequence. Taken together, these observations indicate conformational heterogeneity in the protein-free human U2-U6 snRNA complex consistent with a model in which the RNA has sufficient conformational flexibility to facilitate inter-conversion between steps of splicing in situ.

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“…Conversely, Zacharias and Hagerman (42) observed a decrease in bending for 3U-RNA with an increase in Mg 2+ concentration, whereas our data suggest that 3T-DNA is slightly more bent in the presence of Mg 2+ . This difference could result from specific metal binding to the RNA but not DNA bulge or from stacking or other differences between this DNA and RNA (43,44). The ability of X-ray interferometry to probe beyond structure averages should render this technique particularly valuable in determining the origin of such differences.…”

Section: X-ray Interferometry To Probe the Effects Of Bulge Sequence Andmentioning

confidence: 99%

From a structural average to the conformational ensemble of a DNA bulge

Shi¹,

Beauchamp

Harbury

et al. 2014

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

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Direct experimental measurements of conformational ensembles are critical for understanding macromolecular function, but traditional biophysical methods do not directly report the solution ensemble of a macromolecule. Small-angle X-ray scattering interferometry has the potential to overcome this limitation by providing the instantaneous distance distribution between pairs of gold-nanocrystal probes conjugated to a macromolecule in solution. Our X-ray interferometry experiments reveal an increasing bend angle of DNA duplexes with bulges of one, three, and five adenosine residues, consistent with previous FRET measurements, and further reveal an increasingly broad conformational ensemble with increasing bulge length. The distance distributions for the AAA bulge duplex (3A-DNA) with six different Au-Au pairs provide strong evidence against a simple elastic model in which fluctuations occur about a single conformational state. Instead, the measured distance distributions suggest a 3A-DNA ensemble with multiple conformational states predominantly across a region of conformational space with bend angles between 24 and 85 degrees and characteristic bend directions and helical twists and displacements. Additional X-ray interferometry experiments revealed perturbations to the ensemble from changes in ionic conditions and the bulge sequence, effects that can be understood in terms of electrostatic and stacking contributions to the ensemble and that demonstrate the sensitivity of X-ray interferometry. Combining X-ray interferometry ensemble data with molecular dynamics simulations gave atomic-level models of representative conformational states and of the molecular interactions that may shape the ensemble, and fluorescence measurements with 2-aminopurinesubstituted 3A-DNA provided initial tests of these atomistic models. More generally, X-ray interferometry will provide powerful benchmarks for testing and developing computational methods. helix-junction-helix | SAXSA grand challenge in biology is to understand the complex free-energy landscape of macromolecules and to decipher the resulting conformational ensembles. To perform their biological functions, macromolecules must adopt a multiplicity of conformations. Balancing and controlling different conformational states is central to biological processes including protein folding, allostery and signaling, and the stepwise assembly and function of macromolecular machines. To understand these complex molecules requires characterization of their free-energy landscapes-i.e., their equilibrium conformational ensembles. Precise measurements of conformational ensembles could allow quantitative modeling of the folding and function of biological macromolecules, would provide valuable experimental data to test current computational models and assumptions, and might facilitate the rational design of specifically acting inhibitors (1, 2).Techniques including NMR and EPR relaxation have been developed to incisively probe motions in the ensemble on different time scales, ranging from pic...

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The bulge region of HIV-1 TAR RNA binds metal ions in solution

Cited by 60 publications

References 57 publications

Mg2+-induced Variations in the Conformation and Dynamics of HIV-1 TAR RNA Probed Using NMR Residual Dipolar Couplings

Mg2+-induced Variations in the Conformation and Dynamics of HIV-1 TAR RNA Probed Using NMR Residual Dipolar Couplings

Conformational heterogeneity of the protein-free human spliceosomal U2-U6 snRNA complex

From a structural average to the conformational ensemble of a DNA bulge

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