Mg2+-induced variations in the conformation and dynamics of HIV-1 TAR RNA probed using NMR residual dipolar couplings

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 13C-1H RDCs measured in TAR at [Mg(2+)]:[TAR] stoichiometric ratios of approximately 3:1 (TAR(3.0Mg)) and approximately 4.5:1 (TAR(4.5Mg)) revealed that Mg(2+) reduces the average inter-helical angle from 47(+/-5) degrees in TAR(free) to 5(+/-7) degrees in TAR(4.5Mg). In contrast to the TAR(free) state, the generalized degree of order for the two stems in TAR(4.5Mg) 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.     

Argininamide binding arrests global motions in HIV-1 TAR RNA: comparison with Mg2+-induced conformational stabilization

The structure and dynamics of the stem-loop transactivation response element (TAR) RNA from the human immunodeficiency virus type-1 (HIV-1) bound to the ligand argininamide (ARG) has been characterized using a combination of a large number of residual dipolar couplings (RDCs) and trans-hydrogen bond NMR methodology. Binding of ARG to TAR changes the average inter-helical angle between the two stems from approximately 47 degrees in the free state to approximately 11 degrees in the bound state, and leads to the arrest of large amplitude (+/-46 degrees ) inter-helical motions observed previously in the free state. While the global structural dynamics of TAR-ARG is similar to that previously reported for TAR bound to Mg2+, there are substantial differences in the hydrogen bond alignment of bulge and neighboring residues. Based on a novel H5(C5)NN experiment for probing hydrogen-mediated 2hJ(N,N) scalar couplings as well as measured RDCs, the TAR-ARG complex is stabilized by a U38-A27.U23 base-triple involving an A27.U23 reverse Hoogsteen hydrogen bond alignment as well as by a A22-U40 Watson-Crick base-pair at the junction of stem I. These hydrogen bond alignments are not observed in either the free or Mg2+ bound forms of TAR. The combined conformational analysis of TAR under three states reveals that ligands and divalent ions can stabilize similar RNA global conformations through distinct interactions involving different hydrogen bond alignments in the RNA.     

Concerted motions in HIV-1 TAR RNA may allow access to bound state conformations: RNA dynamics from NMR residual dipolar couplings.

Ground-state dynamics in RNA is a critical precursor for structural adaptation observed ubiquitously in protein-RNA recognition. A tertiary conformational analysis of the stem-loop structural element in the transactivation response element (TAR) from human immunodeficiency virus type 1 (HIV-I) RNA is presented using recently introduced NMR methods that rely on the measurement of residual dipolar couplings (RDC) in partially oriented systems. Order matrix analysis of RDC data provides evidence for inter-helical motions that are of amplitude 46(+/-4) degrees, of random directional character, and that are executed about an average conformation with an inter-helical angle between 44 degrees and 54 degrees. The generated ensemble of TAR conformations have different organizations of functional groups responsible for interaction with the trans-activator protein Tat, including conformations similar to the previously characterized bound-state conformation. These results demonstrate the utility of RDC-NMR for simultaneously characterizing RNA tertiary dynamics and average conformation, and indicate an avenue for TAR complex formation involving tertiary structure capture.     

Constructing RNA dynamical ensembles by combining MD and motionally decoupled NMR RDCs: new insights into RNA dynamics and adaptive ligand recognition

We describe a strategy for constructing atomic resolution dynamical ensembles of RNA molecules, spanning up to millisecond timescales, that combines molecular dynamics (MD) simulations with NMR residual dipolar couplings (RDC) measured in elongated RNA. The ensembles are generated via a Monte Carlo procedure by selecting snap-shot from an MD trajectory that reproduce experimentally measured RDCs. Using this approach, we construct ensembles for two variants of the transactivation response element (TAR) containing three (HIV-1) and two (HIV-2) nucleotide bulges. The HIV-1 TAR ensemble reveals significant mobility in bulge residues C24 and U25 and to a lesser extent U23 and neighboring helical residue A22 that give rise to large amplitude spatially correlated twisting and bending helical motions. Omission of bulge residue C24 in HIV-2 TAR leads to a significant reduction in both the local mobility in and around the bulge and amplitude of inter-helical bending motions. In contrast, twisting motions of the helices remain comparable in amplitude to HIV-1 TAR and spatial correlations between them increase significantly. Comparison of the HIV-1 TAR dynamical ensemble and ligand bound TAR conformations reveals that several features of the binding pocket and global conformation are dynamically preformed, providing support for adaptive recognition via a ‘conformational selection’ type mechanism.     

Structural plasticity and Mg2+ binding properties of RNase P P4 from combined analysis of NMR residual dipolar couplings and motionally decoupled spin relaxation

The P4 helix is an essential element of ribonuclease P (RNase P) that is believed to bind catalytically important metals. Here, we applied a combination of NMR residual dipolar couplings (RDCs) and a recently introduced domain-elongation strategy for measuring "motionally decoupled" relaxation data to characterize the structural dynamics of the P4 helix from Bacillus subtilis RNase P. In the absence of divalent ions, the two P4 helical domains undergo small amplitude (approximately 13 degrees) collective motions about an average interhelical angle of 10 degrees. The highly conserved U7 bulge and helical residue C8, which are proposed to be important for substrate recognition and metal binding, are locally mobile at pico- to nanosecond timescales and together form the pivot point for the collective domain motions. Chemical shift mapping reveals significant association of Mg2+ ions at the P4 major groove near the flexible pivot point at residues (A5, G22, G23) previously identified to bind catalytically important metals. The Mg2+ ions do not, however, significantly alter the structure or dynamics of P4. Analysis of results in the context of available X-ray structures of the RNA component of RNase P and structural models that include the pre-tRNA substrate suggest that the internal motions observed in P4 likely facilitate adaptive changes in conformation that take place during folding and substrate recognition, possibly aided by interactions with Mg2+ ions. Our results add to a growing view supporting the existence of functionally important internal motions in RNA occurring at nanosecond timescales.     

Molecular dynamics studies of the HIV-1 TAR and its complex with argininamide

The dynamic behavior of HIV-1 TAR and its complex with argininamide is investigated by means of molecular dynamics simulations starting from NMR structures, with explicit inclusion of water and periodic boundary conditions particle mesh Ewald representation of the electrostatic energy. During simulations of free and argininamide-bound TAR, local structural patterns, as determined by NMR experiments, were reproduced. An interdomain motion was observed in the simulations of free TAR, which is absent in the case of bound TAR, leading to the conclusion that the free conformation of TAR is intrinsically more flexible than the bound conformation. In particular, in the bound conformation the TAR–argininamide interface is very well ordered, as a result of the formation of a U·A·U base triple, which imposes structural constraints on the global conformation of the molecule. Free energy analysis, which includes solvation contributions, was used to evaluate the influence of van der Waals and electrostatic terms on formation of the complex and on the conformational rearrangement from free to bound TAR.     

Dynamic ensemble view of the conformational landscape of HIV-1 TAR RNA and allosteric recognition

RNA conformational dynamics and the resulting structural heterogeneity play an important role in RNA functions, e.g., recognition. Recognition of HIV-1 TAR RNA has been proposed to occur via a conformational capture mechanism. Here, using ultrafast time-resolved fluorescence spectroscopy, we have probed the complexity of the conformational landscape of HIV-1 TAR RNA and monitored the position-dependent changes in the landscape upon binding of a Tat protein-derived peptide and neomycin B. In the ligand-free state, the TAR RNA samples multiple families of conformations with various degrees of base stacking around the three-nucleotide bulge region. Some subpopulations partially resemble those ligand-bound states, but the coaxially stacked state is below the detection limit. When Tat or neomycin B binds, the bulge region as an ensemble undergoes a conformational transition in a position-dependent manner. Tat and neomycin B induce mutually exclusive changes in the TAR RNA underlying the mechanism of allosteric inhibition at an ensemble level with residue-specific details. Time-resolved anisotropy decay measurements revealed picosecond motions of bases in both ligand-free and ligand-bound states. Mutation of a base pair at the bulge--stem junction has differential effects on the conformational distributions of the bulge bases. A dynamic model of the ensemble view of the conformational landscape for HIV-1 TAR RNA is proposed, and the implication of the general mechanism of RNA recognition and its impact on RNA-based therapeutics are discussed.     

The role of magnesium and potassium ions in the molecular mechanism of ribosome assembly: hydrodynamic, conformational, and thermal stability studies of 16 S RNA from Escherichia coli ribosomes

In an attempt to understand the role of magnesium ion in ribosome assembly in vitro, the hydrodynamic shape, conformation, and thermal stability of ribosomal 16 S RNA were studied systematically as a function of Mg2+ concentration by sedimentation velocity, intrinsic viscosity, circular dichroism, and difference ultraviolet absorption spectroscopy. These results were then compared with the corresponding parameters obtained for 16 S RNA under the optimal conditions of reconstitution, i.e., at 37 degrees C, 20 mM Mg2+, an ionic strength equal to 0.37, and pH 7.8 [S. H. Allen, and K.-P. Wong (1978) J. Biol. Chem. 253, 8759-8766]. When the 360 mM KCl required for reconstitution of 30 S ribosomes is added to the medium, only subtle conformational changes are observed, consistent with the destabilization of the conformation, thus making the RNA molecule more "open" and accessible to protein binding. However, when the concentration of Mg2+ is lowered from 20 to 1 mM, the hydrodynamic parameters indicate that the 16 S RNA is partially unfolded, while thermal denaturation studies suggest that the amount of base-stacking and base-pairing is not concomitantly altered. Further removal of the Mg2+ by dialysis against a pH 7.8 buffer containing no Mg2+ results in a drastic decrease of secondary structure and indicates that the Mg2+ is required for maintenance of the pairing, stacking, and stability of the nucleotide bases, in addition to the long range interactions which result in a compact structure. The results suggest that the 20 mM Mg2+ is required for the 16 S RNA molecules to assume the proper secondary and tertiary structure containing the protein-binding sites, while the high K+ concentration (360 mM KCl) is needed for "loosening up" the RNA, making the protein binding sites more accessible to the ribosomal proteins for molecular recognition and binding as well as for the conformational changes that occur during ribosome assembly.     

Towards structural genomics of RNA: rapid NMR resonance assignment and simultaneous RNA tertiary structure determination using residual dipolar couplings

We report a new residual dipolar couplings (RDCs) based NMR procedure for rapidly determining RNA tertiary structure demonstrated on a uniformly (15)N/(13)C-labeled 27 nt variant of the trans-activation response element (TAR) RNA from HIV-I. In this procedure, the time-consuming nuclear Overhauser enhancement (NOE)-based sequential assignment step is replaced by a fully automated RDC-based assignment strategy. This approach involves examination of all allowed sequence-specific resonance assignment permutations for best-fit agreement between measured RDCs and coordinates for sub-structures in a target RNA. Using idealized A-form geometries to model Watson-Crick helices and coordinates from a previous X-ray structure to model a hairpin loop in TAR, the best-fit RDC assignment solutions are determined very rapidly (相似文献   

Molecular dynamics simulation reveals conformational switching of water-mediated uracil-cytosine base-pairs in an RNA duplex

A 4 ns molecular dynamics simulation of an RNA duplex (r-GGACUUCGGUCC)(2 )in solution with Na+ and Cl- as counterions was performed. The X-ray structure of this duplex includes two water-mediated uracil-cytosine pairs. In contrast to the other base-pairs in the duplex the water-mediated pairs switch between different conformations. One conformation corresponds to the geometry of the water-mediated UC pairs in the duplex X-ray structure with water acting both as hydrogen-bond donor and acceptor. Another conformation is close to that of a water-mediated UC base-pair found in the X-ray structure of the 23 S rRNA sarcin/ricin domain. In this case the oxygen of the water molecule is linked to two-base donor sites. For a very short time also a direct UC base-pair and a further conformation that is similar to the one found in the RNA duplex structure but exhibits an increased H3(U)...N3(C) distance is observed. Water molecules with unusually long residence times are involved in the water-mediated conformations. These results indicate that the dynamic behaviour of the water-mediated UC base-pairs differs from that of the duplex Watson-Crick and non-canonical guanine-uracil pairs with two or three direct hydrogen bonds. The conformational variability and increased flexibility has to be taken into account when considering these base-pairs as RNA building blocks and as recognition motifs.     

NMR studies of RNA dynamics and structural plasticity using NMR residual dipolar couplings

An increasing number of RNAs are being discovered that perform their functions by undergoing large changes in conformation in response to a variety of cellular signals, including recognition of proteins and small molecular targets, changes in temperature, and RNA synthesis itself. The measurement of NMR residual dipolar couplings (RDCs) in partially aligned systems is providing new insights into the structural plasticity of RNA through combined characterization of large-amplitude collective helix motions and local flexibility in noncanonical regions over a wide window of biologically relevant timescales (相似文献   

Conformational dynamics of RNA-peptide binding: a molecular dynamics simulation study

Molecular dynamics simulations of the binding of the heterochiral tripeptide KkN to the transactivation responsive (TAR) RNA of HIV-1 is presented, using an all-atom force field with explicit water. To obtain starting structures for the TAR-KkN complex, semirigid docking calculations were performed that employ an NMR structure of free TAR RNA. The molecular dynamics simulations show that the starting structures in which KkN binds to the major groove of TAR (as it is the case for the Tat-TAR complex of HIV-1) are unstable. On the other hand, the minor-groove starting structures are found to lead to several binding modes, which are stabilized by a complex interplay of stacking, hydrogen bonding, and electrostatic interactions. Although the ligand does not occupy the binding position of Tat protein, it is shown to hinder the interhelical motion of free TAR RNA. The latter is presumably necessary to achieve the conformational change of TAR RNA to bind Tat protein. Considering the time evolution of the trajectories, the binding process is found to be ligand-induced and cooperative. That is, the conformational rearrangement only occurs in the presence of the ligand and the concerted motion of the ligand and a large part of the RNA binding site is necessary to achieve the final low-energy binding state.     

Structural equilibria in RNA as revealed by 19F NMR

We have incorporated 5-fluorouridine into several sites within a 19-mer RNA modelled on the translational operator of the MS2 bacteriophage. The 19F NMR spectra demonstrate the different chemical shifts of helical and loop fluorouridines of the hairpin secondary structure. Addition of salt gives rise to a species in which the loop fluorouridine gains the chemical shift of its helical counterparts, due to the formation of the alternative bi-molecular duplex form. This is supported by UV thermal melting behaviour which becomes highly dependent on the RNA concentration. Distinct 19F NMR signals for duplex and hairpin forms allow the duplex-hairpin equilibrium constant to be determined under a range of conditions, enabling thermodynamic characterisation and its salt dependence to be determined. Mg2+ also promotes duplex formation, but more strongly than Na+, such that at 25 degrees C, 10 mM MgCl2 has a comparable duplex-promoting effect to 300 mM NaCl. A similar effect is observed with Sr2+, but not Ca2+ or Ba2+. Additional hairpin species are observed in the presence of Na+ as well as Mg2+, Ca2+, Sr2+ and Ba2+ ions. The overall, ensemble average, hairpin conformation is therefore salt-dependent. Electrostatic considerations are thus involved in the balance between different hairpin conformers as well as the duplex-hairpin equilibrium. The data presented here demonstrate that 19F NMR is a powerful tool for the study of conformational heterogeneity in RNA, which is particularly important for probing the effects of metal ions on RNA structure. The thermodynamic characterisation of duplex-hairpin equilibria will also be valuable in the development of theoretical models of nucleic acid structure.     

EPR spectroscopic analysis of TAR RNA-metal ion interactions

Metal ion-induced changes in HIV-1 TAR RNA internal dynamics were determined by the changes in EPR spectral width for TAR RNAs containing spin-labeled nucleotides (U23, U25, U38, and U40). This gave a dynamic signature for each of 10 metal ions studied, which fell into one of three distinct groups. While Li(+) and K(+) had little effect on TAR RNA internal dynamics, Na(+) unexpectedly had a dynamic signature that was similar to Ca(2+) and Sr(2+), with a decrease in mobility at U23 and U38, little or no change at U25, and an increase in mobility at U40. Mg(2+), Co(2+), Ni(2+), Zn(2+), and Ba(2+) had similar effects on U23, U38, and U40, but the mobility of U25 was markedly increased. Our results show that RNA dynamics change upon metal binding to the TAR RNA bulge, indicating that RNA structure adapts to accommodate metal ions of different size and coordination properties.     

Influence of the 2'-hydroxyl group conformation on the stability of A-form helices in RNA

The 2'-hydroxyl group plays fundamental roles in both the structure and the function of RNA, and is the major determinant of the conformational and thermodynamic differences between RNA and DNA. Here, we report a conformational analysis of 2'-OH groups of the HIV-2 TAR RNA by means of NMR scalar coupling measurements in solution. Our analysis supports the existence of a network of water molecules spanning the minor groove of an RNA A-form helix, as has been suggested on the basis of a high-resolution X-ray study of an RNA duplex. The 2'-OH protons of the lower stem nucleotides of the TAR RNA project either towards the O3' or towards the base, where the 2'-OH group can favorably participate in H-bonding interactions with a water molecule situated in the nucleotide base plane. We observe that the k(ex) rate of the 2'-OH proton with the bulk solvent anti-correlates with the base-pair stability, confirming the involvement of the 2'-OH group in a collective network of H-bonds, which requires the presence of canonical helical secondary structure. The methodology and conformational analysis presented here are broadly applicable and facilitate future studies aimed to correlate the conformation of the 2'-OH group with both the structure and the function of RNA and RNA-ligand complexes.     

Effect of magnesium ions on the structure of DNA thin films: an infrared spectroscopy study

Utilizing Fourier transform infrared spectroscopy we have investigated the vibrational spectrum of thin dsDNA films in order to track the structural changes upon addition of magnesium ions. In the range of low magnesium concentration ([magnesium]/[phosphate] = [Mg]/[P] < 0.5), both the red shift and the intensity of asymmetric PO₂ stretching band decrease, indicating an increase of magnesium-phosphate binding in the backbone region. Vibration characteristics of the A conformation of the dsDNA vanish, whereas those characterizing the B conformation become fully stabilized. In the crossover range with comparable Mg and intrinsic Na DNA ions ([Mg]/[P] ≈ 1) B conformation remains stable; vibrational spectra show moderate intensity changes and a prominent blue shift, indicating a reinforcement of the bonds and binding in both the phosphate and the base regions. The obtained results reflect the modified screening and local charge neutralization of the dsDNA backbone charge, thus consistently demonstrating that the added Mg ions interact with DNA via long-range electrostatic forces. At high Mg contents ([Mg]/[P] > 10), the vibrational spectra broaden and show a striking intensity rise, while the base stacking remains unaffected. We argue that at these extreme conditions, where a charge compensation by vicinal counterions reaches 92–94%, DNA may undergo a structural transition into a more compact form.     

Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings

We present a protocol for determining the relative orientation and dynamics of A-form helices in 13C/15N isotopically enriched RNA samples using NMR residual dipolar couplings (RDCs). Non-terminal Watson-Crick base pairs in helical stems are experimentally identified using NOE and trans-hydrogen bond connectivity and modeled using the idealized A-form helix geometry. RDCs measured in the partially aligned RNA are used to compute order tensors describing average alignment of each helix relative to the applied magnetic field. The order tensors are translated into Euler angles defining the average relative orientation of helices and order parameters describing the amplitude and asymmetry of interhelix motions. The protocol does not require complete resonance assignments and therefore can be implemented rapidly to RNAs much larger than those for which complete high-resolution NMR structure determination is feasible. The protocol is particularly valuable for exploring adaptive changes in RNA conformation that occur in response to biologically relevant signals. Following resonance assignments, the procedure is expected to take no more than 2 weeks of acquisition and data analysis time.