Structures of two aptamers with differing ligand specificity reveal ruggedness in the functional landscape of RNA
- Yale University, United States
Figures
Figure 1 with 2 supplements
Overall structure of the PRPP riboswitch and its G96A mutant, which is a ppGpp aptamer.
(A) Consensus sequence of the PRPP riboswitch, adapted from Sherlock et al. (Sherlock et al., 2018b). The secondary structure has been updated to show structural information gained from the present …
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Figure 1—source data 1
Summary of fitted binding data without Bmax constraints.
- https://doi.org/10.7554/eLife.36381.006
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Figure 1—source data 2
Raw binding data.
- https://doi.org/10.7554/eLife.36381.007
Figure 1—figure supplement 1
Autoradiograph of a representative PAGE gel from dissociation constant determination for the PRPP aptamer.
The higher of the two bands represents intact PRPP, while the lower band represents degradation products. The leftmost lane is purified PRPP. This leftmost lane represents a sample of PRPP that was …
Figure 1—figure supplement 2
Data from equilibrium dialysis experiments and fits used to calculate dissociation constants.
All experiments were performed in triplicate and the standard deviation for each data point is shown using black error bars. (A) wild type RNA binding to PRPP (purple) and ppGpp (green). The ppGpp …
Figure 2
The binding pocket of the PRPP riboswitch.
(A) Crystal structure of the ligand-binding site in chain A. Relative to Figure 1, the structure is rotated 180° about the y axis. PRPP is depicted as sticks and colored by element with purple …
Figure 3 with 1 supplement
Notable contacts to the PRPP ligand in chain A. PRPP is depicted as sticks and colored by element with purple carbons.
Nucleotides are depicted as sticks and colored by element with blue carbons. Individual nucleotides and metals are labeled. Dashed black lines indicate hydrogen bonds. A dashed green line shows the …
Figure 3—figure supplement 1
Coordination of metals by chain A of the PRPP aptamer.
PRPP is depicted as sticks and colored by element with purple carbons. Nucleotides are depicted as sticks and colored by element with blue carbons. Individual nucleotides and metals are labeled. …
Figure 4 with 1 supplement
The binding pocket of the G96A mutant in complex with ppGpp.
(A) Crystal structure of the ligand-binding site in chain A. ppGpp is depicted as sticks and colored by element with green carbons. Nucleotides are depicted as blue sticks. Metal ions are depicted …
Figure 4—figure supplement 1
A comparison of ppGpp modeled in the syn conformation and the anti conformation.
Chain A is shown. ppGpp has green carbons and is colored by element. The Fo-Fc map was generated using an otherwise complete model lacking ppGpp and is shown contoured at 3.0 σ as a grey mesh.
Figure 5
Notable contacts to the ppGpp ligand in chain A. ppGpp is depicted as sticks with green carbons and is colored by element.
RNA is depicted as sticks with blue carbons and is colored by element. Dashed black lines indicate hydrogen bonds. (A) hydrogen bonds donated from amino groups in C77 and G48 to the 3′-β-phosphate …
Figure 6 with 3 supplements
Comparison of the guanidine binding site in the guanidine aptamer, the M3 binding site in the PRPP aptamer, and the corresponding site in the G96A mutant.
(A) Watson-Crick base pairs with C75 in the wild-type and G96A aptamers. Wild-type and G96A aptamer structures are overlaid. Wild-type RNA is shown as gray sticks. G96A RNA is shown as blue sticks. …
Figure 6—figure supplement 1
Conserved interactions between P1 and P3.
Nucleotides are labeled, depicted as sticks and colored by element with blue carbons. Dashed lines indicate hydrogen bonds. (A) Nucleotides A6 and A68 of the guanidine aptamer. (B) Nucleotides G6 …
Figure 6—figure supplement 2
Predicted model of switching in the tandem guanine-PRPP riboswitch from T. mathranii.
The secondary structure of the RNA is represented as black lines. P0 nucleotides are colored dark red. Guanine aptamer nucleotides that may pair with P0 nucleotides are colored light red. P3 …
Figure 6—figure supplement 3
ppGpp purification and validation.
(A) Anion-exchange chromatography of substrate and product standards. The ppGpp sample standard was purchased from TriLink BioTechnologies. (B) Anion-exchange chromatography of reactions with …
Tables
Table 1
Dissociation constants for PRPP and ppGpp binding to the wild type and G96A T. mathranii aptamers with calculated fold specificity changes.
https://doi.org/10.7554/eLife.36381.011| Dissociation constants for WT and G96A binding to PRPP and ppGpp | ||||
|---|---|---|---|---|
| Construct | Kd for PRPP | Kd for ppGpp | Fold specificity for PRPP over ppGpp | Estimated magnitude of overall specificity switch |
| Wild type | 2.0 ± 0.3 μM | 91 ± 3 μM | 46 | ~40,000 |
| G96A | 1600 ± 200 μM | 1.8 ± 0.1 μM | ~0.001 | |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers |
|---|---|---|---|
| Chemical compound, drug | ppGpp standard | TriLink BioTechnologies | TriLink Biotechnologies: N-6001 |
| Chemical compound, drug | PRPP | Millipore Sigma | Millipore Sigma:P8296- 25MG |
| Commercial assay or kit | Hampton Research Natrix HT Screen | Hampton Research | Hampton Research:HR2- 131 |
| Commercial assay or kit | Harvard Apparatus cassettes | Harvard Apparatus | Harvard Apparatus:742203 |
| Peptide, recombinant protein | Ribose phosphate pyrophosphokinase | Abbexa | Abbexa:abx072019 |
| Sequence-based reagent | T. mathranii genome | NCBI | NCBI:NC_014209.1 |
| Software, algorithm | CCP4 | https://doi.org/10.1107/S0907444910045749 | CCP4:7.0.042; RRID:SCR_007255 |
| Software, algorithm | Coot | https://doi.org/10.1107/S0907444904019158 | Coot:0.8.6.1; RRID:SCR_014222 |
| Software, algorithm | GraphPad | GraphPad | GraphPad:7.0a; RRID:SCR_002798 |
| Software, algorithm | HKL2000 | https://doi.org/10.1016/S0076-6879(97)76066-X | HKL2000:v0.98.714; RRID:SCR_015547 |
| Software, algorithm | Open Source PyMol | SourceForge https://sourceforge.net/projects/pymol/ | PyMol:v1.8.x; RRID:SCR_000305 |
Additional files
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Supplementary file 1
X-ray crystallography statistics from data collection and refinement.
- https://doi.org/10.7554/eLife.36381.019
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Transparent reporting form
- https://doi.org/10.7554/eLife.36381.020
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Reporting file 1
- https://doi.org/10.7554/eLife.36381.021
