Quadruple 9-mer-based protein binding microarray with DsRed fusion protein

Abstract

Background: The interaction between a transcription factor and DNA motif (cis-acting element) is an important regulatory step in gene regulation. Comprehensive genome-wide methods have been developed to characterize protein-DNA interactions. Recently, the universal protein binding microarray (PBM) was introduced to determine if a DNA motif interacts with proteins in a genome-wide manner.

Expression of StMYB1R-1, a Novel Potato Single MYB-Like Domain Transcription Factor, Increases Drought Tolerance

“…Protein DNA-binding properties are traditionally investigated by methods such as EMSAs and filter-binding assays (Garner and Revzin, 1981;Sö derman and Reichard, 1986). The development of the Q9 protein-binding microarray along with the availability of whole-genome sequence information and advances in microarray technology have greatly facilitated our ability to characterize protein DNA-binding specificities in vitro (Kim et al, 2009). The results of Q9 protein-binding microarray analysis and EMSA demonstrated that StMYB1R-1 binds DNA mainly at G / A GATAA sequences ( Fig.…”

“…The quadruple 9-mer (Q9) protein-binding array was developed as a screening tool for rapid identification of DNA-binding sequences of TFs and contains more than 100,000 unique double-stranded DNA oligomers synthesized as quadruples of all possible 9-mer combinations (Kim et al, 2009). Using a DsRed fusion protein of StMYB1R-1 (StMYB1R-1:DsRed) to monitor binding, Q9 proteinbinding microarray analysis yielded 1,028 putative DNA-binding sequences (.3,500 relative signal intensity).…”

“…The StMYB1R-1 fusion protein was purified from Escherichia coli strain BL21-CodonPlus (Stratagene). A total of 200 nM of the StMYB1R-1 protein was incubated with Q9 protein-binding microarray, included 101,073 features from each 9-mers, in phosphate-buffered saline-2% bovine serum albumin, 50 ng/mL salmon testes DNA (Sigma), and 50 mM zinc acetate at 25°C for 1 h (Kim et al, 2009). Fluorescence images were obtained with a 4000B microarray scanner (Molecular Devices).…”

Quadruple 9-mer-Based Protein Binding Microarray Analysis Confirms AACnG as the Consensus Nucleotide Sequence Sufficient for the Specific Binding of AtMYB44

“…We determined the DNA-binding sequence specificity of At-MYB44 by a comprehensive genome-wide method, using a universal PBM (Kim et al, 2009). The PBM was designed to contain quadruples of all possible 9-mer combinations, permitting unequivocal interpretation of the cis-acting elements.…”

“…Using the full-length cDNA of AtMYB44 (TAIR clone 119B8), proteins fused at the N-termini with DsRed fluorescent protein and a polyhistidine-tag were expressed in the Escherichia coli strain BL21-ColonPLus, as described previously (Kim et al, 2009). A protein-binding mixture containing 200 nM fusion protein was incubated with the quadruple 9-mer PBM (Q9-PBM) at 25°C for 1 h. Fluorescence images were obtained with a 4000B microarray scanner (Molecular Devices, USA).…”