(A) Schematic of the activation assay. To measure in vivo activation, we expressed fragments of TF proteins fused to a DNA-binding domain that binds uniquely in the promoter of a genome-integrated …
Data from activation assays.
ADs identified in the activation screen and their overlap with previously-known ADs.
Protein positions are 0-indexed and inclusive of start but exclusive of stop positions.
(A) Distributions of signal from the GFP reporter (in arbitrary units), measured from FACS, in cells expressing artificial TF (aTF) fused to ADs from VP16, Gcn4, or Pho4, or without an AD. Cells …
(A) Tiles were binned by their hydrophobic content and net charge and the median activation of each bin is displayed in color. Activation was strongest for tiles high in both acidic and hydrophobic …
(A) Distribution of hydrophobic content (top) and net charge (bottom) for non-activating tiles (gray) and highly activating tiles (green). Hydrophobicity is computed using the Wimley-White scale and …
(A) Activation of wild-type (orange) and eight scrambled sequences (green) for each of eight ADs. (B) De novo PADDLE predictions (purple) and experimentally measured activation (green) for Arg81 are …
ADs predicted in human and virus proteins and core ADs predicted in yeast.
Protein positions are 0-indexed and inclusive of start but exclusive of stop positions.
(A) Neural network predictions from amino acid composition alone on test data withheld from training. R2 is the coefficient of determination. Compare to PADDLE predictions in Panel B. (B) PADDLE …
(A) To quantify the importance of aa composition, we measured activation of 33 scrambled sequences for each of the 28 strongest 13-aa core ADs (cADs). (Top) Activation of each wild-type cAD divided …
(A) The wild-type-to-scramble ratio of core ADs was not correlated with their wild-type activation strength (Pearson’s r = 0.32, p=0.09). Related to Figure 4A–B. (B) For each position assayed across …
(A) To measure binding in high-throughput, we used mRNA display, expressing our library of TF tiles as a pool of protein fragments covalently tagged with their mRNA sequences (left), and using this …
Data from pull-down assays and list of Med15-binding domains.
(A) (Left) A 4–20% polyacrylamide gel showing in vivo biotinylated GST-TEV-Avi-Med15K123-6xHis protein (denoted Med15) eluted from NiNTA resin (lane 2), and the flow-through (lane 3) and bound beads …
(A) We used a Rosetta peptide docking algorithm (Raveh et al., 2011) to build structural models of the 28 13-aa core ADs (cADs) described above (Figure 4A–D) interacting with two activator-binding …
List of all Med15 ABD and core AD interactions modeled using FlexPepDock.
Structural models of Med15 ABD and core AD interactions generated by FlexPepDock.
The 10 best-scoring models from different binding poses are provided as pdb files. Top500.txt contains the score, cluster number, and RMSD to best model for the 500 best-scoring models.
(A) The best-scoring model of the Pdr1 cAD (blue) bound to the KIX domain (gray). KIX residues important for binding the Pdr1 AD are shown in red (Thakur et al., 2008). The cAD contacts the …
(A) We took 47 pairs of adjacent cADs and measured their activation enhancement factors—the activation of both cADs in tandem divided by the product of activation by each cAD individually. For 40 …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (S. cerevisiae) | BY4711 | ATCC | 200873 | MATalpha trp1delta63 |
Cell line (human) | HEK293T | ATCC | CRL-3216 | |
Sequence-based reagent | Oligo pool libraries | Twist Bioscience | Custom synthesis | Sequences provided in source data files |
Recombinant DNA reagent | pMVS142-pACT1-mCherry-Zif268-EBD-MCS-KAN | Addgene | 99049 | Barak Cohen |
Recombinant DNA reagent | pMVS102-P3-GFP-NATMX6 | Addgene | 99048 | Barak Cohen |
Chemical compound, drug | Fetal bovine serum | Millipore | TMS-031-B | |
Strain, strain background (Escherichia coli) | XL10 Gold Ultracompetent Cells | Agilent | 200315 | For cloning |
Peptide, recombinant protein | Phusion polymerase | New England Biolabs | M0531L | |
Commercial assay or kit | AMPure XP beads | Beckman Coulter | A63880 | |
Recombinant DNA reagent | pFN26A BIND plasmid | Promega | E1380 | |
Recombinant DNA reagent | pGL4.35 plasmid | Promega | E1370 | |
Commercial assay or kit | Dual-Glo Luciferase assay | Promega | E2920 | |
Recombinant DNA reagent | pBirAcm | Avidity | AVB99 | |
Strain, strain background (Escherichia coli) | BL21 Star (DE3) competent cells | ThermoFisher | C601003 | For protein expression |
Commercial assay or kit | Ni-NTA Suerflow resin | Qiagen | 30410 | |
Recombinant DNA reagent | pFASTBac1 plasmid | ThermoFisher | 10360014 | |
Commercial assay or kit | MEGAscript T7 transcription kit | ThermoFisher | AM1333 | |
Peptide, recombinant protein | T4 DNA ligase | New England Biolabs | M0202S | |
Other | Amicon Ultracel 0.5 mL 30K MWCO column | Millipore Sigma | UFC503024 | |
Commercial assay or kit | Model 422 Electro-Eluter | Bio Rad | 1652976 | |
Commercial assay or kit | Retic Lysate IVT Kit | ThermoFisher | AM1200 | |
Sequence-based reagent | PF30P oligo | IDT | Custom synthesis | /5Phos/AA AAA AAA AAA AAA AAA AAA A/iSp9//iSp9//iSp9/AC C/3Puro/ |
Peptide, recombinant protein | SuperScript II Reverse Transcriptase | ThermoFisher | 18064014 | |
Commercial assay or kit | Dynabeads MyOne Streptavidin T1 | ThermoFisher | 65602 | |
Sequence-based reagent | Salmon Sperm DNA | ThermoFisher | 15632011 | |
Peptide, recombinant protein | BSA | New England Biolabs | B9000S | |
Software, algorithm | PADDLE | This paper | github.com/asanborn/PADDLE |