Genetically tunable frustration controls allostery in an intrinsically disordered transcription factor
- Johns Hopkins University, United States
- University of Houston, United States
Figures
Figure 1 with 1 supplement
Transcriptional activity and DNA-binding affinity are not correlated among GR translational isoforms.
Transcriptional activity, monitored by dual luciferase reporter assay (Figure 1—figure supplement 1a and b''), and binding affinity to DNA, monitored by fluorescence anisotropy change (Figure …
Figure 1—figure supplement 1
Transcriptional activity and DNA-binding affinity of GR translational isoforms.
(a) Luciferase assay dosage curves for the constitutively active constructs of the eight GR translational isoforms. Per 30,000 cells, a constant 40 ng of GRE-driven luciferase vector was …
Figure 2 with 1 supplement
Coupling of the R-domain and DBD to the functional F-domain in GR.
(a) TMAO-induced folding for the F-domain alone and with either the R-domain or the DBD. (b) Protease sensitivity assay: comparing F-domain and F-domain with R-domain (left) performed at a protein …
Figure 2—figure supplement 1
Domain stabilities determined by TMAO-induced protein folding transitions.
(a) Thermodynamic parameters obtained from TMAO-induced folding experiments demonstrate that DBD stabilizes a folded conformation of the F-domain while the R-domain destabilizes that conformation. …
Figure 3
Coupling between the R-domain and DBD in GR.
(a) Schematic representation of competitive transfection assay design for constructs of the R-domains of isoforms A, B, C1, and C2 as well as the internally truncated R-domain linked to GR DBD or …
Figure 4
Competing thermodynamic coupling in GR produces frustration.
(a) Schematic view of the thermodynamic configuration of GR. According to this convention, the positive (+) signs between the DBD and F-domain, and the DBD and R-domain signify they are positively …
Figure 5 with 1 supplement
The ensemble allosteric model (EAM) quantitatively describes GR transcriptional activation.
(a) EAM for GR A and C3 isoforms. (b) The model recapitulates the DNA-binding affinity and relative transcriptional activity of A, B, C1, C2, and C3 isoforms. Error bars on experimental data …
Figure 5—figure supplement 1
Probability distribution of each thermodynamic parameter from EAM.
Two independent methods were used to estimate parameters for the EAM from experimental data. The first method was a ‘brute force’ grid search for parameter combinations that satisfied the …
Figure 6 with 1 supplement
Contiguous surface mediates coupling between the DBD and the R-domain.
(a) Schematic representation of the constructs used to identify residues on DBD involved in coupling to the R-domain. (b) EAM predicted changes in binding affinity for mutations that affect the …
Figure 6—figure supplement 1
Mutagenesis used to identify the residues in the DBD that mediate the coupling to the R-domain.
(a) Sequence conservation analysis of the DBD among the GRs in different species and among all members in the steroid hormone receptor family. Above is the secondary structure annotation of the GR …
Additional files
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Supplementary file 1
Mathematica notebook for data fitting.
- https://doi.org/10.7554/eLife.30688.013
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Transparent reporting form
- https://doi.org/10.7554/eLife.30688.014
