Sequence-directed variations in the canonical DNA double helix structure that retain Watson-Crick base-pairing play important roles in DNA recognition, topology, and nucleosome positioning. By using nuclear magnetic resonance relaxation dispersion spectroscopy in concert with steered molecular dynamics simulations, we have observed transient sequence-specific excursions away from Watson-Crick base-pairing at CA and TA steps inside canonical duplex DNA towards low-populated and short-lived A•T and G•C Hoogsteen base-pairs. The observation of Hoogsteen base-pairs in DNA duplexes specifically bound to transcription factors and in damaged DNA sites implies that the DNA double helix intrinsically codes for excited state Hoogsteen base-pairs as a means of expanding its structural complexity beyond that which can be achieved based on Watson-Crick base-pairing. The methods presented here provide a new route for characterizing transient low-populated nucleic acid structures, which we predict will be abundant in the genome and constitute a second transient layer of the genetic code.
RNAs fold into three-dimensional (3D) structures that subsequently undergo large, functionally important, conformational transitions in response to a variety of cellular signals. RNA structures are believed to encode spatially tuned flexibility that can direct transitions along specific conformational pathways. However, this hypothesis has proved difficult to examine directly because atomic movements in complex biomolecules cannot be visualized in 3D by using current experimental methods. Here we report the successful implementation of a strategy using NMR that has allowed us to visualize, with complete 3D rotational sensitivity, the dynamics between two RNA helices that are linked by a functionally important trinucleotide bulge over timescales extending up to milliseconds. The key to our approach is to anchor NMR frames of reference onto each helix and thereby directly measure their dynamics, one relative to the other, using 'relativistic' sets of residual dipolar couplings (RDCs). Using this approach, we uncovered super-large amplitude helix motions that trace out a surprisingly structured and spatially correlated 3D dynamic trajectory. The two helices twist around their individual axes by approximately 53 degrees and 110 degrees in a highly correlated manner (R = 0.97) while simultaneously (R = 0.81-0.92) bending by about 94 degrees. Remarkably, the 3D dynamic trajectory is dotted at various positions by seven distinct ligand-bound conformations of the RNA. Thus even partly unstructured RNAs can undergo structured dynamics that directs ligand-induced transitions along specific predefined conformational pathways.
Using a domain elongation strategy, we decoupled internal motions in RNA from overall rotational diffusion. This allowed us to site-specifically resolve a manifold of motional modes in two regulatory RNAs from HIV-1 with the use of nuclear magnetic resonance spin relaxation methods. Base and sugar librations vary on a picosecond time scale and occur within helical domains that move collectively at diffusion-limited nanosecond time scales. Pivot points are short, functionally important, and highly mobile internal loops. These spontaneous changes in RNA conformation correlate quantitatively with those that follow adaptive recognition of diverse targets. Thus, ligands may stabilize existing RNA conformations rather than inducing new ones.
1. Introduction 3721.1 Residual dipolar couplings as a route to structure and dynamics 3721.2 A brief history of oriented phase high resolution NMR 3742. Theoretical treatment of dipolar interactions 3762.1 Anisotropic interactions as probes of macromolecular structure and dynamics 3762.1.1 The dipolar interaction 3762.1.2 Averaging in the solution state 3772.2 Ordering of a rigid body 3772.2.1 The Saupe order tensor 3782.2.2 Orientational probability distribution function 3802.2.3 The generalized degree of order 3802.3 Molecular structure and internal dynamics 3813. Inducing molecular order in high resolution NMR 3833.1 Tensorial interactions between the magnetic field and anisotropic magnetic susceptibilities 3833.2 Dilute liquid crystal media: a tunable source of order 3843.2.1 Bicelles : from membrane mimics to aligning media 3853.2.2 Filamentous phage 3873.2.3 Transfer of alignment from ordered media to macromolecules 3883.3 Magnetic field alignment 3893.3.1 Paramagnetic assisted alignment 3893.3.2 Advantages of using magnetic alignment 3894. The measurement of residual dipolar couplings 3914.1 Introduction 3914.2 Frequency based methods 3924.2.1 Coupling enhanced pulse schemes 3924.2.2 In phase anti-phase methods (IPAP): 1DNH couplings in proteins 3934.2.3 Exclusive correlated spectroscopy (E-COSY): 1DNH, 1DNC′ and 2DHNC′ 3954.2.4 Extraction of splitting values from the frequency domain 3964.3 Intensity based experiments 3974.3.1 J-Modulated experiments: the measurement of 1DCαHα in proteins 3974.3.2 Phase modulated methods 3994.3.3 Constant time COSY – the measurement of DHH couplings 3994.3.4 Systematic errors in intensity based experiments 4005. Interpretation of residual dipolar coupling data 4015.1 Structure determination protocols utilizing orientational constraints 4015.1.1 The simulated annealing approach 4015.1.2 Order matrix analysis of dipolar couplings 4025.1.3 A discussion of the two approaches 4025.2 Reducing orientational degeneracy 4035.2.1 Multiple alignment media in the simulated annealing approach 4045.2.2 Multiple alignment media in the order matrix approach 4055.3 Simplifying effects arising due to molecular symmetry 4065.4 Database approaches for determining protein structure 4076. Applications to the characterization of macromolecular systems 4086.1 Protein structure refinement 4086.2 Protein domain orientation 4096.3 Oligosaccharides 4136.4 Biomolecular complexes 4156.5 Exchanging systems 4167. Acknowledgements 4188. References 419Within its relatively short history, nuclear magnetic resonance (NMR) spectroscopy has managed to play an important role in the characterization of biomolecular structure. However, the methods on which most of this characterization has been based, Nuclear Overhauser Effect (NOE) measurements for short-range distance constraints and scalar couplings measurements for torsional constraints, have limitations (Wüthrich, 1986). For extended structures, such as DNA helices, for example, propagation of errors in the short distance constraints derived from NOEs leaves the relative orientation of remote parts of the structures poorly defined. Also, the low density of observable protons in contact regions of molecules held together by factors other than hydrophobic packing, leads to poorly defined structures. This is especially true in carbohydrate containing complexes where hydrogen bonds often mediate contacts, and in multi-domain proteins where the area involved in domain–domain contact can also be small. Moreover, most NMR based structural applications are concerned with the characterization of a single, rigid conformer for the final structure. This can leave out important mechanistic information that depends on dynamic aspects and, when motion is present, this can lead to incorrect structural representations. This review focuses on one approach to alleviating some of the existing limitations in NMR based structure determination: the use of constraints derived from the measurement of residual dipolar couplings (D).
The visualization of RNA conformational changes has provided fundamental insights into how regulatory RNAs carry out their biological functions. The RNA structural transitions that have been characterized to date involve long-lived species that can be captured by structure characterization techniques. Here, we report the Nuclear Magnetic Resonance visualization of RNA transitions towards invisible ‘excited states’ (ES), which exist in too little abundance (2–13%) and for too short periods of time (45–250 μs) to allow structural characterization by conventional techniques. Transitions towards ESs result in localized rearrangements in base-pairing that alter building block elements of RNA architecture, including helix-junction-helix motifs and apical loops. The ES can inhibit function by sequestering residues involved in recognition and signaling or promote ATP-independent strand exchange. Thus, RNAs do not adopt a single conformation, but rather exist in rapid equilibrium with alternative ESs, which can be stabilized by cellular cues to affect functional outcomes.
The measurement of residual dipolar couplings in weakly aligned proteins can potentially provide unique information on their structure and dynamics in the solution state. The challenge is to extract the information of interest from the measurements, which normally reflect a convolution of the structural and dynamic properties. We discuss here a formalism which allows a first order separation of their effects, and thus, a simultaneous extraction of structural and motional parameters from residual dipolar coupling data. We introduce some terminology, namely a generalized degree of order, which is necessary for a meaningful discussion of the effects of motion on residual dipolar coupling measurements. We also illustrate this new methodology using an extensive set of residual dipolar coupling measurements made on (15)N,(13)C-labeled human ubiquitin solvated in a dilute bicelle solution. Our results support a solution structure of ubiquitin which on average agrees well with the X-ray structure (Vijay-Kumar, et al., J. Mol. Biol. 1987, 194, 531--544) for the protein core. However, the data are also consistent with a dynamic model of ubiquitin, exhibiting variable amplitudes, and anisotropy, of internal motions. This work suggests the possibility of primary use of residual dipolar couplings in characterizing both structure and anisotropic internal motions of proteins in the solution state.
Rare tautomeric and anionic nucleobases are believed to play fundamental biological roles but their prevalence and functional importance has remained elusive because they exist transiently, in low-abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-populated Watson-Crick like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10−3-10−5) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases.
Preface Conformational changes involving coding and non-coding RNAs form the basis for genetic regulatory elements and provide an important source of complexity for driving many fundamental processes of life. While RNA is highly flexible, the underlying dynamics are robust and limited to transitions between the few conformations that preserve favorable base-pairing and stacking interactions. The mechanisms by which cellular processes harness RNA’s intrinsic dynamic behavior and steer it towards functionally productive pathways are complex. Versatile functions and ease of integration into a wide variety of genetic circuits and biochemical pathways suggests a general and fundamental role for RNA dynamics in cellular processes.
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