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

Abstract

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 (<ms), the measurement of residual dipolar couplings (RDCs) in partially oriented systems 13,14 has opened a new avenue for characterizing the conformational dynamics of flexible biomolecules using NMR. [15][16][17][18][19][20] We recently reported the RDC-NMR characterization of a divalent cation free transactivation response element (TAR) RNA variant (TAR(free)) (Figure 1(a)). 21 The TAR domain has been the subject of intense structural studies, [22][23][24][25] Although several studies have reported evidence for binding of divalent metal ions to TAR, discrepancies persist regarding the effects of divalent ions on the TAR average conformation while little is also known about the effects of divalent ions on TAR dynamics. Using electric birefringence, Zacharias and Hageraman previously reported that Mg 2+ (~2 mM) induces ~50% reduction in the inter-helical angle of a 120 nt long RNA containing the bulge region of TAR. In contrast, a 1.3 Å X-ray structure of an RNA duplex comprising the bulge region of TAR determined in the presence of much higher divalent ion concentrations (...

Argininamide Binding Arrests Global Motions in HIV-1 TAR RNA: Comparison with Mg2+-induced Conformational Stabilization

“…As shown in Figure 1(b) and (c), the agreement between the two sets of measurements is excellent, both in the absence (Figure 1(b)) and presence of phage (Figure 1(c)). The root-mean-square-deviation (RMSD) is substantially smaller than the magnitude of measured RDCs (range between −38.4 Hz and 40.7 Hz) and correlation coefficients R 2 are close to ideal (Figure 1 To elucidate the global structure and dynamics of TAR-ARG, RDCs measured in stems I and II were independently subjected to an order matrix analysis 44,45 using idealized A-form geometries as input coordinates for the two stems as previously described for TAR-FREE 28 and TAR-Mg. 29 As shown in Table 1, the measured RDCs are in very good agreement with the local geometry of idealized A-form helices. The RMSD between measured RDCs and values calculated using best-fit order tensors for stems I (3.9 Hz) and stem II (3.5 Hz) are only slightly larger than the uncertainty in RDC measurements (2.6 Hz).…”

“…The RMSD between measured RDCs and values calculated using best-fit order tensors for stems I (3.9 Hz) and stem II (3.5 Hz) are only slightly larger than the uncertainty in RDC measurements (2.6 Hz). This argues that like in the case of TAR-FREE 28 and TAR-Mg, 29 the local conformation of the two stems in TAR-ARG are accurately modeled using idealized A-form helices.…”

“…[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.…”

“…More recently, we demonstrated using RDC NMR methodology that binding of Mg 2+ to TAR (TAR-Mg) leads to coaxial alignment of the two stems and a solution conformation that is similar to its X-ray structure counterpart determined previously in the presence of divalent ions, 38 and the complete quenching of inter-helical motions. 29 To further explore how the global structure and dynamics of TAR changes in response to recognition, we employed RDCs along with trans-hydrogen bond NMR methodology [39][40][41] to investigate the conformation of TAR bound to ARG. Binding of TAR to ARG was first examined by recording 2D 13 C-1 H correlation spectra of uniformly 13 C-/ 15 N-labeled TAR with increasing ARG concentration.…”

Measuring single-molecule nucleic acid dynamics in solution by two-color filtered ratiometric fluorescence correlation spectroscopy

“…The stabilization effect of magnesium ion on RNA structure is well documented, and, generally, it stabilizes tertiary structure and slows down intramolecular motion (31,34,35). This effect has been clearly measured in the experiment.…”