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 interhelical angle between the two stems from ~47° in the free state to ~11° in the bound state, and leads to the arrest of large amplitude (±46°) 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 Mg 2+ , there are substantial differences in the hydrogen bond alignment of bulge and neighboring residues. Based on a novel H5(C5)NN experiment for probing hydrogenmediated 2h J(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 Mg 2+ 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. Keywords recognition; adaptation; collective motions; NMR; residual dipolar couplingsThere is now ample evidence indicating that many RNAs do not fold into a single welldefined structure, but rather exist as an ensemble of interconverting conformations. [21][22][23] By being uniquely sensitive to motional averaging over a wide window of time scales (picoseconds-milliseconds), the measurement of RDCs has also emerged as a powerful approach for probing the amplitudes and directions of collective motions in biomolecules. [24][25][26][27] We recently employed RDCs to investigate the conformational dynamics of human immunodefiency virus type 1 (HIV-1) transactivation response element (TAR) RNA ( Figure 1(a)). 28,29 The TAR domain has been the subject of numerous investigations because its interaction with the transactivator protein (Tat) is critical for HIV-1 viral replication, 30 rendering it a potential target for therapeutic development. 31 Previous structural studies had established that binding to peptide mimics of Tat, including the ligand argininamide (ARG), induces a change in the TAR global conformation 32-37 from a bent to coaxial interhelical alignment. However little information was available regarding global motions in TAR and its potential role in recognition. Our RDC-NMR study of TAR in the divalent ion free state (TAR-FREE) 28 provided evidence that the two helices undergo large amplitude (±46°) rigid-body collective motions about an average inter-helical angle of 47°. Because the coaxially aligned bound TAR conformations appeared to be dynamically accessible in the free state, these resu...
We examined how static and dynamic deviations from the idealized A-form helix propagate into errors in the principal order tensor parameters determined using residual dipolar couplings (rdcs). A 20-ns molecular dynamics (MD) simulation of the HIV-1 transactivation response element (TAR) RNA together with a survey of spin relaxation studies of RNA dynamics reveals that pico-to-nanosecond local motions in non-terminal Watson-Crick base-pairs will uniformly attenuate base and sugar one bond rdcs by approximately 7%. Gaussian distributions were generated for base and sugar torsion angles through statistical comparison of 40 RNA X-ray structures solved to <3.0 A resolution. For a typical number (>or=11) of one bond C-H base and sugar rdcs, these structural deviations together with rdc uncertainty (1.5 Hz) lead to average errors in the magnitude and orientation of the principal axis of order that are <9% and <4 degrees, respectively. The errors decrease to <5% and <4 degrees for >or=17 rdcs. A protocol that allows for estimation of error in A-form order tensors due to both angular deviations and rdc uncertainty (Aform-RDC) is validated using theoretical simulations and used to analyze rdcs measured previously in TAR in the free state and bound to four distinct ligands. Results confirm earlier findings that the two TAR helices undergo large changes in both their mean relative orientation and dynamics upon binding to different targets.
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 (
Approaches developed thus for extracting structural and dynamical information from RDCs have rested on the assumption that motions do not affect molecular alignment. However, it is well established that molecular alignment in ordered media is dependent on conformation, and slowly interconverting conformational substates may exhibit different alignment properties. Neglecting these correlation effects can lead to aberrations in the structural and dynamical analysis of RDCs and diminish the utility of RDCs in probing motions between domains having similar alignment propensities. Here, we introduce a new approach based on measurement of magnetic field induced residual dipolar couplings in nucleic acids which can explicitly take into account such correlations and demonstrate measurements of motions between two "magnetically equivalent" domains in the transactivation response element (TAR) RNA.
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