Allosteric mechanism of the V. vulnificus adenine riboswitch resolved by four-dimensional chemical mapping
- Stanford University, United States
Figures
Figure 1 with 3 supplements
Models of the add riboswitch.
(A–B) Three-state models for conformational changes and ligand binding in the add riboswitch. In current models, there is an apoB state (left). This state is OFF (Shine-Dalgarno sequence and AUG …
Figure 1—figure supplement 1
One-dimensional SHAPE profiles of add riboswitch constructs.
Three different constructs were probed by SHAPE: (top) a 71-nt aptamer only construct (nts 13–83); (middle) a 113-nt construct (nts 13–125) including the expression platform, matching the previous …
Figure 1—figure supplement 2
Mutate-and-map (M2) experiments on the add riboswitch.
Measurements were acquired in 10 mM MgCl2, 50 mM Na-HEPES, pH 8.0, in (A) the absence of adenine ligand and (B) in the presence of ligand (5 mM adenine).
Figure 1—figure supplement 3
Candidate alternative helices from M2 analyses.
(A) Helices modeled to be in the adenine riboswitch based on M2 data with and without adenine, using prior analysis assuming a dominant secondary structure. To estimate uncertainties, simulated data …
Figure 2 with 1 supplement
Mutate-map-rescue (M2R) enables inference of helix frequencies.
(A) Interrogation of base pairs by compensatory mutagenesis and chemical mapping, illustrated on a four-way junction (nts 126–235) of the E. coli 16S ribosomal RNA. Experiments involve ‘quartets’ of …
Figure 2—figure supplement 1
Correlation between rescue factor and helix frequency from Rfam simulations of mutate-map-rescue (M2R).
(A) Boxplots showing helix frequency vs. rescue factor, over all simulated helices. Purple lines mark median, open boxes cover 25th to 75th percentile, cyan whiskers show 5th and 95th percentile, …
Figure 3 with 2 supplements
Experimental data and frequency estimates for helices P1 and P4B in the add riboswitch structural ensembles.
Top: two example secondary structures showing sequences of the add riboswitch and helices probed. The presented structures are for illustration of the helices only, and do not imply co-existence in …
Figure 3—figure supplement 1
All 188 single base-pair M2R quartets for add riboswitch.
Helices P1 (7 bp), P2 (6 bp), P3 (6 bp), P5 (6 bp), P1B (5 bp), P2B (2 bp), P4A (4 bp), P4B (4 bp), P4C (2 bp), P6 (5 bp), P8 (4 bp), P9 (5 bp), P10 (3 bp), P11 (9 bp), P12 (3 bp), P13 (4 bp), P14 …
Figure 3—figure supplement 2
All 82 double base-pair M2R quartets for add riboswitch.
Helices P1, P2, P3, P5, P1B, P2B, P4A, P4B, P4C, P6, and P13 were tested for both no adenine and 5 mM adenine conditions. MutP2 from a previous study (Reining et al., 2013) is also included. Rescue …
Figure 4
Summary of helix frequencies inferred for the add riboswitch.
Posterior distributions over helix frequencies (A) without adenine ligand and (B) with 5 mM adenine ligand. Curves have been smoothed through kernel density estimation. Sub-panels separate helices …
Figure 5 with 2 supplements
Double-base-pair mutations to lock each helix of the adenine riboswitch.
One-dimensional SHAPE reactivity of candidate constructs for locking each add riboswitch helix. In each candidate, two consecutive base pairs of the helix were switched to alternative Watson-Crick …
Figure 5—figure supplement 1
All double-base-pair mutations tested as possible locking mutations.
One-dimensional SHAPE reactivity of candidate constructs for locking each add riboswitch helix. The MutP2 variant is derived from reference (Reining et al., 2013); it shows less SHAPE increase in …
Figure 5—figure supplement 2
Multi-probe chemical mapping of stabilizers and linear fitting of P1 and P4A.
(A) Five-modifier (SHAPE, 1M7, glyoxal, terbium (III), UV 302 nm) reactivity profile for lock-P1 and lock-P4A constructs. χ2 score-based linear fitting yields apoA state as 48% (SHAPE), 58% (DMS), …
Figure 6 with 2 supplements
Anticorrelation between P1 and P4B helices in ligand-free add riboswitch structural ensemble, inferred through lock-mutate-map-rescue (M2R).
(A–C) M2R quartets probing P1 in the context of lock-P4B mutations show no rescue. (D–F) M2R quartets probing P4B in the context of lock-P1 mutations show no rescue. Panels (A,D) show possible …
Figure 6—figure supplement 1
All 82 single base-pair LM2R quartets for add riboswitch.
Helices P2 and P4B are tested in context of lock-P1 stabilizing mutations; helices P1, P4A, and P4B are tested in lock-P2 stabilizing mutations; helices P2, P1B, P2B, and P4B are tested in context …
Figure 6—figure supplement 2
All 15 single base-pair LM2R quartets for add riboswitch with the MutP2 construct.
Helices P1, P4A, and P4B were tested under no adenine condition. Rescue factor for each quartet is given in the title and colored as in Figure 2—figure supplement 1.
Figure 7 with 1 supplement
Correlation analysis for functionally important helix-helix pairs in the ligand-free add riboswitch structural ensemble.
Posterior probability distributions, smoothed through kernel density estimation, for correlation of (A-C) P1 and P4B, based on LM2R experiments (A) locking P4B and probing P1, (B) locking P1 and …
Figure 7—figure supplement 1
Posterior probability distributions over helix frequencies and helix-helix correlations for individual LM2R experiments.
In each panel, top two sub-panels show posterior probability distributions, smoothed through kernel density estimation, over helix frequency, estimated from the experimental M2R rescue factors in …
Figure 8 with 3 supplements
Current model for the add riboswitch structural ensemble.
The proposed model favors a Monod-Wyman-Changeux (population shift, conformational selection) model of allostery. The ligand-free apoB state (left) sequesters the Shine-Dalgarno sequence and AUG …
Figure 8—figure supplement 1
Simulation of temperature dependence on switching efficiency.
Thermodynamic parameters used are: ΔHKpre = 52 kJ.mol−1, ΔSKpre = 167 J.mol−1.K−1, ΔHKd = 238 kJ.mol−1, ΔSKd = 110 J.mol−1.K−1. Ligand concentrations: [Llow]=0.01 μM; [Lhigh]=1.0 μM for (top) panels …
Figure 8—figure supplement 2
Models of additional structures that the add riboswitch can form, based on mutate-and-map on selected single mutant backgrounds.
Single mutants (A) A109U, (B) G78C, (C) A116U, (D) G44C, (E) G37C, and (F) G81C were selected from a clustering analysis of WT M2 data. Models are for dominant structures of the RNA based on …
Figure 8—figure supplement 3
Higher-order mutate-and-map on selected single mutant backgrounds.
(A) A109U, (B) G78C, (C) A116U, (D) G44C, (E) G37C, and (F) G81C. A comparison of 1D SHAPE profile to WT under both no adenine and 5 mM adenine conditions are shown along with complete M2 datasets; …
Appendix 1—figure 1
Base-pair-wise classification of M2R and LM2R quartets by the 4-bin auto-score.
Table summary of 4-bin classification by human expert and automatic algorithm on in vitro (A) single base-pair M2R, (B) double base-pair M2R, and (C) LM2R and MutP2 are shown. Each symbol represents …
Appendix 1—figure 2
M2R quartets and helices tested for P4-P6 and GIR1.
(A) M2R quartets of P4-P6 domain testing P5C, alt-P5C, and P6. Rescue factor for each quartet is given in the title and colored as in Figure S1. (B–C) Secondary structures of P4-P6 and GIR1 …
Appendix 1—figure 3
Performance of 4-bin auto-score classifier.
Histogram of in silico (A) training data (16S-FWJ, P4P6, GIR1, Hox, PB2; total of 162 quartets), (B) test data (add single and double base-pair M2R; total of 242 quartets), and (C) Rfam family …
Tables
Table 1
Helix frequency estimates for single base-pair M2R, double base-pair M2R, LM2R experiments.
Median helix frequencies from Rfam simulations corresponding to each experimentally observed rescue factor are reported. The experimentally observed rescue factor for each helix was averaged across …
| Wild type* | In locked contexts† | ||||
|---|---|---|---|---|---|
| [adenine] | 0 mM | 5 mM | 0 mM | ||
| P1 | 53% | 79% | Lock P1 | P2 | 42% |
| P2 | 31% | 64% | P4B | 4% | |
| P3 | 73% | 56% | Lock P2 | P1 | 76% |
| P5 | 75% | 29% | P4A | 4% | |
| P1B | 44% | 3% | P4B | 4% | |
| P2B | 17% | 17% | Lock P4A | P1B | 9% |
| P4A | 48% | 4% | P2B | 6% | |
| P4B | 55% | 4% | P2 | 6% | |
| P4C | 68% | 4% | P4B | 55% | |
| P6 | 6% | 5% | Lock P1B | P2B | 53% |
| P8 | 9% | 4% | P4A | 55% | |
| P9 | 12% | 3% | P4B | 80% | |
| P10 | 3% | 3% | P3 | 82% | |
| P11 | 12% | 16% | Lock P4B | P1B | 8% |
| P12 | 10% | 6% | P2B | 4% | |
| P13 | 9% | 4% | P1 | 5% | |
| P14 | 4% | 4% | P2 | 6% | |
| P15 | 12% | 36% | P4A | 47% | |
| P16 | 14% | 8% | Mut P2 | P1 | 38% |
| P17 | 4% | 4% | P4A | 8% | |
| P4B | 13% | ||||
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*Median helix frequencies inferred from mutate-map-rescue, compensatory rescue read out by chemical mapping across the transcript.
†Lock-mutate-map-rescue, mutate-map-rescue carried out in the context of mutations that 'lock' the specified helices.
Table 2
Helix-helix correlation estimates from LM2R experiments.
Median values are reported. Full posterior distributions are presented in main text Figure 6 and Figure 7—figure supplement 1.
| Helix-helix | Correlation value |
|---|---|
| P1-P4B | 0.052 |
| P2-P4B | 0.069 |
| P2-P4A | 0.089 |
| P1-P2 | 1.315 |
| P4A-P4B | 0.902 |
| P1B-P4A | 0.44 |
| P1B-P4B | 0.534 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.29602.026
