(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 …
Summary of fitted binding data without Bmax constraints.
Raw binding data.
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 …
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 …
(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 …
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 …
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. …
(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 …
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.
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 …
(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. …
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 …
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 …
(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 …
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 |
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 |
X-ray crystallography statistics from data collection and refinement.