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Proc Natl Acad Sci U S A. 2018 May 22;115(21):E4796-E4805. doi: 10.1073/pnas.1722055115. Epub 2018 May 4.

Systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria.

Author information

1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
2
Department of Physics, California Institute of Technology, Pasadena, CA 91125.
3
Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, 751 24 Uppsala, Sweden.
4
Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA 91125.
5
Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.
6
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125; phillips@pboc.caltech.edu.
7
Department of Applied Physics, California Institute of Technology, Pasadena, CA 91125.

Abstract

Gene regulation is one of the most ubiquitous processes in biology. However, while the catalog of bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in these genomes are regulated. At present, characterizing the molecular mechanisms by which individual regulatory sequences operate requires focused efforts using low-throughput methods. Here, we take a first step toward multipromoter dissection and show how a combination of massively parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used to dissect multiple bacterial promoters in a systematic way. We show this approach on both well-studied and previously uncharacterized promoters in the enteric bacterium Escherichia coli In all cases, we recover nucleotide-resolution models of promoter mechanism. For some promoters, including previously unannotated ones, the approach allowed us to further extract quantitative biophysical models describing input-output relationships. Given the generality of the approach presented here, it opens up the possibility of quantitatively dissecting the mechanisms of promoter function in E. coli and a wide range of other bacteria.

KEYWORDS:

DNA affinity chromatography; gene regulation; mass spectrometry; massively parallel reporter assay; quantitative models

PMID:
29728462
DOI:
10.1073/pnas.1722055115
Free PMC Article

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