High-throughput analysis of protein-DNA binding affinity.
Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro.
Genomics Unit, Centro Nacional de Biotecnología-CSIC, Madrid, Spain.
José M
JM
Franco-Zorrilla
Roberto
R
Solano
D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002846:Chromatography, Affinity; D018744:DNA, Plant / Q000378:metabolism*; D004926:Escherichia coli; D057166:High-Throughput Screening Assays; D058619:Maltose-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D018515:Plant Leaves / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014026:Nicotiana / Q000378:metabolism; D059467:Transcriptome
D029681:Arabidopsis Proteins; D018744:DNA, Plant; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins