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Characterization of Bacterial Transcriptional Regulatory Networks in Escherichia coli through Genome-Wide In Vitro Run-Off Transcription/RNA-seq (ROSE)

The global characterization of transcriptional regulatory networks almost exclusively uses in vivo conditions, thereby providing a snapshot on multiple regulatory interactions at the same time. To complement these approaches, we developed and applied a method for characterizing bacterial promoters g...

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Autores principales: Schmidt, Pascal, Brandt, David, Busche, Tobias, Kalinowski, Jörn
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10301672/
https://www.ncbi.nlm.nih.gov/pubmed/37374890
http://dx.doi.org/10.3390/microorganisms11061388
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author Schmidt, Pascal
Brandt, David
Busche, Tobias
Kalinowski, Jörn
author_facet Schmidt, Pascal
Brandt, David
Busche, Tobias
Kalinowski, Jörn
author_sort Schmidt, Pascal
collection PubMed
description The global characterization of transcriptional regulatory networks almost exclusively uses in vivo conditions, thereby providing a snapshot on multiple regulatory interactions at the same time. To complement these approaches, we developed and applied a method for characterizing bacterial promoters genome-wide by in vitro transcription coupled to transcriptome sequencing specific for native 5′-ends of transcripts. This method, called ROSE (run-off transcription/RNA-sequencing), only requires chromosomal DNA, ribonucleotides, RNA polymerase (RNAP) core enzyme, and a specific sigma factor, recognizing the corresponding promoters, which have to be analyzed. ROSE was performed on E. coli K-12 MG1655 genomic DNA using Escherichia coli RNAP holoenzyme (including σ70) and yielded 3226 transcription start sites, 2167 of which were also identified in in vivo studies, and 598 were new. Many new promoters not yet identified by in vivo experiments might be repressed under the tested conditions. Complementary in vivo experiments with E. coli K-12 strain BW25113 and isogenic transcription factor gene knockout mutants of fis, fur, and hns were used to test this hypothesis. Comparative transcriptome analysis demonstrated that ROSE could identify bona fide promoters that were apparently repressed in vivo. In this sense, ROSE is well-suited as a bottom-up approach for characterizing transcriptional networks in bacteria and ideally complementary to top-down in vivo transcriptome studies.
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spelling pubmed-103016722023-06-29 Characterization of Bacterial Transcriptional Regulatory Networks in Escherichia coli through Genome-Wide In Vitro Run-Off Transcription/RNA-seq (ROSE) Schmidt, Pascal Brandt, David Busche, Tobias Kalinowski, Jörn Microorganisms Article The global characterization of transcriptional regulatory networks almost exclusively uses in vivo conditions, thereby providing a snapshot on multiple regulatory interactions at the same time. To complement these approaches, we developed and applied a method for characterizing bacterial promoters genome-wide by in vitro transcription coupled to transcriptome sequencing specific for native 5′-ends of transcripts. This method, called ROSE (run-off transcription/RNA-sequencing), only requires chromosomal DNA, ribonucleotides, RNA polymerase (RNAP) core enzyme, and a specific sigma factor, recognizing the corresponding promoters, which have to be analyzed. ROSE was performed on E. coli K-12 MG1655 genomic DNA using Escherichia coli RNAP holoenzyme (including σ70) and yielded 3226 transcription start sites, 2167 of which were also identified in in vivo studies, and 598 were new. Many new promoters not yet identified by in vivo experiments might be repressed under the tested conditions. Complementary in vivo experiments with E. coli K-12 strain BW25113 and isogenic transcription factor gene knockout mutants of fis, fur, and hns were used to test this hypothesis. Comparative transcriptome analysis demonstrated that ROSE could identify bona fide promoters that were apparently repressed in vivo. In this sense, ROSE is well-suited as a bottom-up approach for characterizing transcriptional networks in bacteria and ideally complementary to top-down in vivo transcriptome studies. MDPI 2023-05-25 /pmc/articles/PMC10301672/ /pubmed/37374890 http://dx.doi.org/10.3390/microorganisms11061388 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Schmidt, Pascal
Brandt, David
Busche, Tobias
Kalinowski, Jörn
Characterization of Bacterial Transcriptional Regulatory Networks in Escherichia coli through Genome-Wide In Vitro Run-Off Transcription/RNA-seq (ROSE)
title Characterization of Bacterial Transcriptional Regulatory Networks in Escherichia coli through Genome-Wide In Vitro Run-Off Transcription/RNA-seq (ROSE)
title_full Characterization of Bacterial Transcriptional Regulatory Networks in Escherichia coli through Genome-Wide In Vitro Run-Off Transcription/RNA-seq (ROSE)
title_fullStr Characterization of Bacterial Transcriptional Regulatory Networks in Escherichia coli through Genome-Wide In Vitro Run-Off Transcription/RNA-seq (ROSE)
title_full_unstemmed Characterization of Bacterial Transcriptional Regulatory Networks in Escherichia coli through Genome-Wide In Vitro Run-Off Transcription/RNA-seq (ROSE)
title_short Characterization of Bacterial Transcriptional Regulatory Networks in Escherichia coli through Genome-Wide In Vitro Run-Off Transcription/RNA-seq (ROSE)
title_sort characterization of bacterial transcriptional regulatory networks in escherichia coli through genome-wide in vitro run-off transcription/rna-seq (rose)
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10301672/
https://www.ncbi.nlm.nih.gov/pubmed/37374890
http://dx.doi.org/10.3390/microorganisms11061388
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