Real-time multidimensional NMR follows RNA folding with second resolution

Abstract

Conformational transitions and structural rearrangements are central to the function of many RNAs yet remain poorly understood. We have used ultrafast multidimensional NMR techniques to monitor the adenine-induced folding of an adenine-sensing riboswitch in real time, with nucleotide-resolved resolution. By following changes in 2D spectra at rates of approximately 0.5 Hz, we identify distinct steps associated with the ligand-induced folding of the riboswitch. Following recognition of the ligand, long range loop-loop interactions form and are then progressively stabilized before the formation of a fully stable complex over approximately 2-3 minutes. The application of these ultrafast multidimensional NMR methods provides the opportunity to determine the structure of RNA folding intermediates and conformational trajectories.

Fig. 1.

Sequence and secondary structures of the ligand-free and ligand-bound adenine-sensing riboswitch ligand-binding domain of the add A-riboswitch from Vibrio vulnificus (10). The secondary structure of the free riboswitch is based on NMR data (11, 12), as well as the assignments reported in this study.

Fig. 2.

Two-dimensional spectra of the ligand-free and ligand-bound adenine-sensing riboswitch aptamer domain. Conventional ¹H, ¹⁵N-HSQCs of the free (A) and bound (B) ¹⁵N-G-labeled RNA. Conventional ¹H, ¹⁵N-HSQCs of the free (C) and bound (D) ¹⁵N-U-labeled RNA. UltraSOFAST ¹H, ¹⁵N correlation spectra of the free (E) and bound (F) ¹⁵N-U-labeled RNA.

Fig. 3.

Signal buildup and decay curves for representative sites in the riboswitch; different spectra are averaged together in the three set of data, corresponding to (left to right) time resolutions of 2.4, 4.8 and 9.6 sec. Plots cover the first 200 sec of the reaction and markers on the right of each plot denote the statistical noise spread (95% confidence limits) of each measurement.

Fig. 4.

(A) Representative real-time 2D HMQC NMR spectra recorded at pH 6.1 and 298 K on a ∼1.7 mM ¹⁵N-G-labeled adenine riboswitch ligand-binding domain; the times indicated in each frame correspond to the time point following addition of adenine and Mg²⁺ to the free RNA solution. (B) Representative real-time 2D HMQC spectra of the [¹⁵N-U]-labeled riboswitch recorded at the indicated times following ligand addition in the magnet by rapid mixing. Spectral assignments are indicated on the figures.

Fig. 5.

Secondary structure representation of the ligand-induced folding of the adenine-sensing riboswitch ligand-binding domain, as revealed by real-time 2D NMR. Dotted lines and zigzag symbols indicate unstable hydrogen bonding and flexible structural features, respectively. (A) Free conformation of the riboswitch; helices P2 and P3 are formed, but P1 is only partially stable, as also observed by single molecule measurements (14); (B) Formation of the ligand-binding pocket occurs rapidly, with a rate (16 sec) comparable to the 1D NMR observation (12), but helix P1 remains partially unfolded (14); (C) Tertiary contacts between loop 2 and 3 are observed after the formation of the ligand-binding pocket; and (D) they are fully stabilized after approximately 1 min, though structural flexibility is retained in helix P1. (E) Formation of the final bound structure is completed after 2–3 min.