High-Throughput Affinity Measurements of Transcription Factor and DNA Mutations Reveal Affinity and Specificity Determinants

被引:17
|
作者
Aditham, Arjun K. [1 ,2 ]
Markin, Craig J. [3 ]
Mokhtari, Daniel A. [3 ]
DelRosso, Nicole [4 ]
Fordyce, Polly M. [1 ,2 ,5 ,6 ]
机构
[1] Stanford Univ, Dept Bioengn, Stanford, CA 94305 USA
[2] Stanford Univ, Stanford ChEM H, Stanford, CA 94305 USA
[3] Stanford Univ, Dept Biochem, Stanford, CA 94305 USA
[4] Stanford Univ, Grad Program Biophys, Stanford, CA 94305 USA
[5] Stanford Univ, Dept Genet, Stanford, CA 94305 USA
[6] Chan Zuckerberg Biohub, San Francisco, CA 94110 USA
基金
美国国家科学基金会; 加拿大健康研究院;
关键词
AMINO-ACID SUBSTITUTIONS; FACTOR-BINDING-SITES; DOUBLE-MUTANT CYCLES; CRYSTAL-STRUCTURE; MICROFLUIDIC AFFINITY; SYSTEMS-APPROACH; SEQUENCE; PHO4; EXPRESSION; PREDICTION;
D O I
10.1016/j.cels.2020.11.012
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to 210 mutants of a model yeast TF (Pho4) interacting with 9 oligonucleotides (>1,800 K(d)s) 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.
引用
收藏
页码:112 / +
页数:27
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