Abstract
Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for ≈65% of promoters we remain ignorant of their regulation. Until we crack this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method, Reg-Seq, that links massively parallel reporter assays with mass spectrometry to produce a base pair resolution dissection of more than a E. coli promoters in 12 growth conditions. We demonstrate that the method recapitulates known regulatory information. Then, we examine regulatory architectures for more than 80 promoters which previously had no known regulatory information. In many cases, we also identify which transcription factors mediate their regulation. This method clears a path for highly multiplexed investigations of the regulatory genome of model organisms, with the potential of moving to an array of microbes of ecological and medical relevance.
Highlights
DNA sequencing is as important to biology as the telescope is to astronomy
The method introduced here provides a framework for solving the problem of deciphering the regulatory genome by connecting perturbation and response, mapping information flow from individual nucleotides in a promoter sequence to downstream gene expression, determining how much information each promoter base pair carries about the level of gene expression
By using Reg-Seq on these genes, we were able to demonstrate that this method recovers what was already known about binding sites of transcription factors for well-characterized promoters (Appendix 2—figures 2 and 3), and whether there are any important differences between the results of the methods presented here and the previous generation of experiments based on fluorescence and cell-sorting as a readout of gene expression (Kinney et al, 2010; Belliveau et al, 2018)
Summary
We are living in the age of genomics, where DNA sequencing has become cheap and routine. Despite these incredible advances, how all of this genomic information is regulated and deployed remains largely enigmatic. We demonstrate how we can infer the logical and regulatory interactions that control bacterial decision making by tapping into the power of DNA sequencing as a biophysical tool. The method introduced here provides a framework for solving the problem of deciphering the regulatory genome by connecting perturbation and response, mapping information flow from individual nucleotides in a promoter sequence to downstream gene expression, determining how much information each promoter base pair carries about the level of gene expression
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