High-Throughput Affinity Measurements of Transcription Factor and DNA Mutations Reveal Affinity and Specificity Determinants
| Autor: | Arjun K. Aditham, Nicole DelRosso, Craig J. Markin, Polly M. Fordyce, Daniel A. Mokhtari |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0303 health sciences
Histology Basic helix-loop-helix Oligonucleotide Chemistry Mutant Pho4 Cell Biology Computational biology DNA Pathology and Forensic Medicine 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Mutation Transcriptional regulation Humans Binding site Transcription factor 030217 neurology & neurosurgery 030304 developmental biology Transcription Factors |
| Zdroj: | Cell systems. 12(2) |
| ISSN: | 2405-4720 |
| Popis: | Transcription factors (TFs) bind regulatory DNA to control gene expression, and mutations to either TFs or DNA can alter binding affinities to rewire regulatory networks and drive phenotypic variation. While studies have profiled energetic effects of DNA mutations extensively, we lack similar information for TF variants. Here, we present STAMMP (simultaneous transcription factor affinity measurements via microfluidic protein arrays), a high-throughput microfluidic platform enabling quantitative characterization of hundreds of TF variants simultaneously. Measured affinities for ∼210 mutants of a model yeast TF (Pho4) interacting with 9 oligonucleotides (>1,800 Kds) reveal that many combinations of mutations to poorly conserved TF residues and nucleotides flanking the core binding site alter but preserve physiological binding, providing a mechanism by which combinations of mutations in cis and trans could modulate TF binding to tune occupancies during evolution. Moreover, biochemical double-mutant cycles across the TF-DNA interface reveal molecular mechanisms driving recognition, linking sequence to function. A record of this paper's Transparent Peer Review process is included in the Supplemental Information. |
| Databáze: | OpenAIRE |
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