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 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 either or both states are highly conserved. 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.

Graphical Abstract

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 of SL3 distal domain show new resonances that appear at elevated temperatures, colored with solid gray bars and dashed gray line; (D) 2D ¹H–¹H NOESY spectra at 5°C (blue) and 25°C (black) of imino region colored with a gray bar in panel C; (E) 2D ¹H–¹³C HSQC spectrum of adenine C2H2 resonances shows over 20 resonances compared to the expected 16 resonances, indicative of slow exchange between multiple states.

Figure 2.

DMS-MaPseq and DREEM clustering supports the presence of a two-state ensemble. (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) SL3e and SL3a secondary structure models 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 shows two-state conformational ensemble of SL3 in in vitro full-length 7SK RNA. (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 (nts 210–264). (B) DREEM clustering shows SL3a and SL3e states with populations that are consistent with the SL3 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, with number labels colored green or red for corresponding SL3a- or SL3e-state resonance assignments. (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 for SL3 distal (black), SL3a-top (green), and SL3e-top (purple) shows SL3 distal construct is a combination of SL3a and SL3e states.

Figure 5.

Comparison of SL3e and SL3a populations from NMR and DREEM analysis. (A) ¹H–¹⁵N imino HSQC spectrum of SL3 distal at 5°C. Labels for P1 stem resonances are colored dark blue, SL3e state resonances are colored purple, SL3a state resonances are colored green, and P2 stem resonances that are not in exchange are colored light blue. (B) Plot of weighted average chemical shift perturbations of SL3e- and SL3a-state imino resonances of SL3 distal in panel A. Solid bars indicate imino resonances that are only observed in the SL3e (purple) or SL3a (green) states. (C) NMR and DREEM analysis of SL3 distal show excellent agreement in the determined populations of SL3e (purple) and SL3a (green) states at 25°C and 37°C.

Figure 6.

Point substitutions lock SL3 into single conformer. Secondary structure models of SL3 distal constructs. (A) E-lock and (B) A-lock with mutations indicated in red. (C) DMS-MaPseq profile of E-lock shown as bar plot. Red bar indicates point substitution, purple bars indicate reporter residues identified in Figure 2D. (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 the SL3e state. SL3a resonance labels are colored green. Asterisks next to U223 and U250 resonances indicate perturbed and new chemical shift, respectively, due to C224A substitution. (F) DMS-MaPseq profile of A-lock shown as bar plot. Green bars indicate reporter residues identified in Figure 2D. (G) Correlation plot of DMS-MaPseq data of A-lock and SL3 distal construct SL3a cluster shows excellent agreement. (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 the SL3a state. SL3e resonance labels are colored purple. (I) ¹H–¹³C 2D HSQC of adenine C2H2 resonances of SL3 distal (black), E-lock (purple), and A-lock (green). New A224 C2H2 resonance is labeled in red with an asterisk. (J) Thermal unfolding profiles of SL3 distal (black), E-lock (purple) and A-lock (green) constructs.

Figure 7.

Linear combination analysis validates a two-state physical model. DMS-MaPseq profiles of (A) SL3 distal construct and (B) SL3 region of full-length 7SK RNA show excellent agreement to a linear combination of SL3e and SL3a states, with populations consistent with those determined using DREEM. DMS-MaPseq mutational fraction values colored black, fit line colored red and residual colored gray.

Figure 8.

7SK SL3 sequence conservation and two-state exchange model. (A) Sequence conservation plotted on SL3e and SL3a states using R2R (53) shows conservation of both potential states. (B) Proposed model of exchange between SL3e and SL3a states. Residues that are involved in base-pair rearrangements are shown in purple (SL3e), red (where SL3e base-pairs are indicated with purple dashed lines and SL3a base-pairs are indicated with green dashed lines), and green (SL3a).