Structures of two aptamers with differing ligand specificity reveal ruggedness in the functional landscape of RNA

6 figures, 2 tables and 3 additional files

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 …

https://doi.org/10.7554/eLife.36381.003
Figure 1—source data 1

Summary of fitted binding data without Bmax constraints.

https://doi.org/10.7554/eLife.36381.006
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 …

https://doi.org/10.7554/eLife.36381.004
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 …

https://doi.org/10.7554/eLife.36381.005
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 …

https://doi.org/10.7554/eLife.36381.008
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 …

https://doi.org/10.7554/eLife.36381.009
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. …

https://doi.org/10.7554/eLife.36381.010
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 …

https://doi.org/10.7554/eLife.36381.012
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.

https://doi.org/10.7554/eLife.36381.013
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 …

https://doi.org/10.7554/eLife.36381.014
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. …

https://doi.org/10.7554/eLife.36381.015
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 …

https://doi.org/10.7554/eLife.36381.016
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 …

https://doi.org/10.7554/eLife.36381.017
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 …

https://doi.org/10.7554/eLife.36381.018

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
ConstructKd for PRPPKd for ppGppFold specificity for PRPP over ppGppEstimated magnitude of overall specificity switch
Wild type2.0 ± 0.3 μM91 ± 3 μM46~40,000
G96A1600 ± 200 μM1.8 ± 0.1 μM~0.001
Key resources table
Reagent type (species) or
resource
DesignationSource or referenceIdentifiers
Chemical compound,
drug
ppGpp standardTriLink BioTechnologiesTriLink Biotechnologies:
N-6001
Chemical compound,
drug
PRPPMillipore SigmaMillipore Sigma:P8296-
25MG
Commercial assay or
kit
Hampton Research
Natrix HT Screen
Hampton ResearchHampton Research:HR2-
131
Commercial assay or
kit
Harvard Apparatus
cassettes
Harvard ApparatusHarvard Apparatus:742203
Peptide, recombinant
protein
Ribose phosphate
pyrophosphokinase
AbbexaAbbexa:abx072019
Sequence-based
reagent
T. mathranii genomeNCBINCBI:NC_014209.1
Software, algorithmCCP4https://doi.org/10.1107/S0907444910045749CCP4:7.0.042;
RRID:SCR_007255
Software, algorithmCoothttps://doi.org/10.1107/S0907444904019158Coot:0.8.6.1;
RRID:SCR_014222
Software, algorithmGraphPadGraphPadGraphPad:7.0a;
RRID:SCR_002798
Software, algorithmHKL2000https://doi.org/10.1016/S0076-6879(97)76066-XHKL2000:v0.98.714;
RRID:SCR_015547
Software, algorithmOpen Source PyMolSourceForge https://sourceforge.net/projects/pymol/PyMol:v1.8.x;
RRID:SCR_000305

Additional files

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