Genomic SELEX Screening of Regulatory Targets of Escherichia coli Transcription Factors

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1837; p. 49
• Main Authors: Shimada, Tomohiro, Ogasawara, Hiroshi, Ishihama, Akira
• Format: Journal Article
• Language: English
• Published: United States 2018
• Subjects: Aptamers, Nucleotide; Computational Biology - methods; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Gene Expression Regulation, Bacterial; Genome, Bacterial; Genomic Library; Genomics - methods; High-Throughput Screening Assays; Recombinant Fusion Proteins; Regulatory Sequences, Ribonucleic Acid; SELEX Aptamer Technique; Transcription Factors - genetics; Transcription Factors - isolation & purification; Transcription Factors - metabolism; Transcription, Genetic; Transcription factor: Sigma factor; Genomic SELEX; RNA polymerase; DNA-protein interaction; Escherichia coli; Regulatory target
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-8675-0_4

The genome of Escherichia coli K-12 is transcribed by a single species of RNA polymerase. The selectivity of its transcriptional targets is modulated via two-steps of protein-protein interaction: at the first step, seven species of the sigma subunit are involved, at the second step, a total of approximately 300 species of transcription factor (TFs). For the identification of the regulatory targets of these two groups of regulatory proteins, we developed two in vitro approaches, "Genomic SELEX" (currently designated as gSELEX) and "PS (promoter-specific)-TF" screenings. Here, we describe a detailed protocol of the genomic SELEX screening system which uses purified regulatory proteins and fragments of genomic DNA from E. coli.