Visualizing spatially correlated dynamics that directs RNA conformational transitions

Abstract

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.

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

“…This task is extremely challenging even for simple HJH motifs, which represent the building blocks of complex nucleic acids structure and an ideal starting point for developing such approaches (13,45). The average structural information from single-molecule FRET experiments with bulged DNAs (31) was reproduced by X-ray interferometry ( Fig.…”

“…Compared with other NMR techniques, RDCs provide long-range angular structure information that helps to generate equilibrium ensemble models (9). In combination with molecular dynamic simulations, RDCs have been used to generate ensemble models for small disordered proteins (7, 10), DNA duplexes (11), and a RNA bulge motif (12)(13)(14). In addition to RDCs, relaxation dispersion and paramagnetic relaxation enhancement have been used to detect and characterize conformational states that are in low abundance in an ensemble (5).…”

Structural basis and dynamics of multidrug recognition in a minimal bacterial multidrug resistance system

“…Thus, the backbone of the binding pocket is flexible on the micro-to millisecond time scale before antibiotic binding, but becomes locked when the pocket is filled in the complex. The motions in the microsecond to millisecond time scale of residues located in the cleft of apo TipAS favor conformational selection (64,65) over an induced fit mechanism for the antibiotic recognition. Accordingly, helices α11 and α12 of the TipAS·antibiotic complexes exhibit the largest displacements upon ligand binding (Fig.…”

Structure of a low-population binding intermediate in protein-RNA recognition

“…1 and Fig. S1) with a highly dynamic structure (10,12,13). The NMR structures of free TAR (14)(15)(16) and of TAR bound to peptide fragments of Tat and to peptide mimetics of Tat in HIV (16)(17)(18)(19)(20) and other lentiviruses (21,22) revealed the conformational properties of TAR in its free and bound states, and demonstrated that this RNA molecule undergoes significant dynamic rearrangements associated with its functions.…”

“…To address this problem, we focused on the well-studied process by which HIV, like other lentiviruses, hijacks the host transcription machinery to activate transcription of the viral genome (9)(10)(11)(12)(13). In HIV, transactivation (Fig.…”