Genome-wide Functional Characterization of ?Escherichia coli Promoters and Sequence Elements Encoding Their Regulation

Using a novel multiplexed reporter assay, we characterize promoter activity of hundreds of thousands of DNA sequences spanning the entire E. coli genome. We use this powerful assay to identify promoters throughout the E. coli genome and systematically dissect their regulatory motifs which encode promoter activity using a series of experiments. Overall design: 1) We measure promoter activity of 17,798 reported transcription start sites (TSSs) and find that a majority of reported TSSs are likely due to transcriptional noise, rather than productive transcription. 2) We measure promoter activity of 321,057 DNA fragments spanning both strands of the E. coli genome with 8.5x coverage in rich LB and minimal glucose media. 3) We measure promoter activity of synthesized fragments overlapping promoter regions identified by characterizing genomic fragments. 4) We perform scanning mutagenesis of 2,142 tss-associated promoters and measure promoter activity of all mutagenized variants.