Visualizing a two-state conformational ensemble in stem-loop 3 of the transcriptional regulator 7SK RNA

Abstract

Structural plasticity is integral to RNA function; however, there are currently few methods to quantitatively resolve RNAs that have multiple structural states. NMR spectroscopy is a powerful approach for resolving conformational ensembles but is size-limited. Chemical probing is well-suited for large RNAs but provides limited structural and no kinetics information. Here, we integrate the two approaches to visualize a two-state conformational ensemble for the central stem-loop 3 (SL3) of 7SK RNA, a critical element for 7SK RNA function in transcription regulation. We find that the SL3 distal end exchanges between two equally populated yet structurally distinct states in both isolated SL3 constructs and full-length 7SK RNA. We rationally designed constructs that lock SL3 into a single state and demonstrate that both chemical probing and NMR data fit to a linear combination of the two states. Comparison of vertebrate 7SK RNA sequences shows conservation of both states, suggesting functional importance. These results provide new insights into 7SK RNA structural dynamics and demonstrate the utility of integrating chemical probing with NMR spectroscopy to gain quantitative insights into RNA conformational ensembles.

Keywords: RNA dynamics; chemical mapping; conformational exchange; phylogenetic analysis; solution NMR spectroscopy.

Figure 1.

7SK RNA SL3 construct design and NMR evidence of conformational exchange a) Cartoon schematic of 7SK RNA secondary structure; b) SL3 distal domain (nts 210–264) constructs for NMR studies; c) 1D ¹H imino proton NMR spectra show new resonances appear at elevated temperatures; d) 2D ¹H-¹H NOESY spectra show differences at 5 °C and 25 °C; e) 2D ¹H-¹³C HSQC spectrum of adenine C2H2 resonances shows greater than the expected number of resonances indicative of slow exchange between multiple states.

Figure 2.

DMS-MaPseq and DREEM clustering supports the presence of two conformers. a) Average mutational fraction of SL3 distal construct. Residues are colored by nucleotide (A, red; C, blue; G, yellow; U, green). b-c) mutational fraction after DREEM clustering. d) Secondary structure models of SL3e (left), SL3a (center), and difference plot (right) colored according to DMS-MaPseq reactivity shows key reporter residues in the P2 stem and J2/J3 loop. e) Secondary structure model of SL3e, colored by motif (P1 dark blue, J1/2 green, P2 light blue, J2/3 yellow, P3 red). Inset: Schematic of differences between SL3e and SL3a secondary structures

Figure 3.

DMS-MaPseq study of in vitro full-length 7SK RNA shows two-state conformational exchange. a) top: average mutational fraction for full-length 7SK RNA, bottom: zoom-in panel of SL3 distal end region corresponding to the isolated domain construct. b) DREEM clusters of SL3a and SL3e conformers are consistent with the domain construct.

Figure 4.

Subdomain constructs of the SL3 top stem lock SL3 into a single state. a) Secondary structures of SL3a-top and SL3e-top constructs. Constructs are colored by secondary structure motifs in SL3e as seen in Figure 2e. b) ¹H-¹H NOESY spectrum of imino region of SL3a-top (green) and SL3e-top (purple) constructs. c) ¹H-¹⁵N 2D HSQC spectrum of uridine N3H3 resonances. d) Plot of weighted average chemical shift perturbations of SL3e and SL3a imino resonances in SL3 distal construct.

Figure 5.

Point substitutions lock SL3 into single conformer. Secondary structure models of SL3 distal constructs a) E-lock and b) A-lock. c) E-lock DMS-MaPseq profile shown as bar plot. Red bar indicates point substitution, purple bars indicate reporter residues identified in Figure 2. DREEM clustering shows 100% SL3e conformer. d) Correlation plot of DMS-MaPseq data of E-lock and SL3 distal construct SL3e cluster show excellent agreement. e) ¹H-¹⁵N 2D HSQC of uridine imino resonances of SL3 distal (black) and E-lock (purple) show good agreement with superimposition of resonances corresponding to SL3e conformer. f) DMS-MaPseq profile of A-lock shown as bar plot. Green bars indicate reporter residues identified in Figure 2. DREEM clustering shows 100% SL3a conformer. g) Correlation plot of DMS-MaPseq data of A-lock and SL3 distal construct SL3a cluster show excellent agreement. Open circle indicates outlier residue A219. h) ¹H-¹⁵N 2D HSQC of uridine imino resonances of SL3 distal (black) and A-lock (green) show good agreement with superimposition of resonances corresponding to SL3a conformer. i) ¹H-¹³C 2D HSQC of adenine C2H2 resonances of SL3 distal (black), E-lock (purple), and A-lock (green). j) Thermal unfolding profiles of SL3 distal (black), E-lock (purple), and A-lock (green).

Figure 6.

Linear combination analysis fits a two-state physical model. a) SL3 distal construct and b) SL3 region of full-length 7SK RNA. DMS-MaPseq mutational fraction values colored black, fit line colored red, and residual colored gray.

Figure 7.

7SK SL3 sequence conservation and exchange model. a) Sequence conservation plotted on SL3e and SL3a states using R2R shows conservation of both potential states. b) Model of exchange between SL3e and SL3a states. Residues that are involved in base-pair rearrangements are shown in purple (SL3e), red (intermediate), and green (SL3a).