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Identification and characterization of preferred DNA-binding sites for the Thermus thermophilus transcriptional regulator FadR

One of the primary transcriptional regulators of fatty acid homeostasis in many prokaryotes is the protein FadR. To better understand its biological function in the extreme thermophile Thermus thermophilus HB8, we sought to first determine its preferred DNA-binding sequences in vitro using the combi...

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Autores principales: Lee, Minwoo, Um, Hyejin, Van Dyke, Michael W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597230/
https://www.ncbi.nlm.nih.gov/pubmed/28902898
http://dx.doi.org/10.1371/journal.pone.0184796
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author Lee, Minwoo
Um, Hyejin
Van Dyke, Michael W.
author_facet Lee, Minwoo
Um, Hyejin
Van Dyke, Michael W.
author_sort Lee, Minwoo
collection PubMed
description One of the primary transcriptional regulators of fatty acid homeostasis in many prokaryotes is the protein FadR. To better understand its biological function in the extreme thermophile Thermus thermophilus HB8, we sought to first determine its preferred DNA-binding sequences in vitro using the combinatorial selection method Restriction Endonuclease Protection, Selection, and Amplification (REPSA) and then use this information to bioinformatically identify potential regulated genes. REPSA determined a consensus FadR-binding sequence 5´-TTRNACYNRGTNYAA-3´, which was further characterized using quantitative electrophoretic mobility shift assays. With this information, a search of the T. thermophilus HB8 genome found multiple operons potentially regulated by FadR. Several of these were identified as encoding proteins involved in fatty acid biosynthesis and degradation; however, others were novel and not previously identified as targets of FadR. The role of FadR in regulating these genes was validated by physical and functional methods, as well as comparative genomic approaches to further characterize regulons in related organisms. Taken together, our study demonstrates that a systematic approach involving REPSA, biophysical characterization of protein-DNA binding, and bioinformatics can be used to postulate biological roles for potential transcriptional regulators.
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spelling pubmed-55972302017-09-15 Identification and characterization of preferred DNA-binding sites for the Thermus thermophilus transcriptional regulator FadR Lee, Minwoo Um, Hyejin Van Dyke, Michael W. PLoS One Research Article One of the primary transcriptional regulators of fatty acid homeostasis in many prokaryotes is the protein FadR. To better understand its biological function in the extreme thermophile Thermus thermophilus HB8, we sought to first determine its preferred DNA-binding sequences in vitro using the combinatorial selection method Restriction Endonuclease Protection, Selection, and Amplification (REPSA) and then use this information to bioinformatically identify potential regulated genes. REPSA determined a consensus FadR-binding sequence 5´-TTRNACYNRGTNYAA-3´, which was further characterized using quantitative electrophoretic mobility shift assays. With this information, a search of the T. thermophilus HB8 genome found multiple operons potentially regulated by FadR. Several of these were identified as encoding proteins involved in fatty acid biosynthesis and degradation; however, others were novel and not previously identified as targets of FadR. The role of FadR in regulating these genes was validated by physical and functional methods, as well as comparative genomic approaches to further characterize regulons in related organisms. Taken together, our study demonstrates that a systematic approach involving REPSA, biophysical characterization of protein-DNA binding, and bioinformatics can be used to postulate biological roles for potential transcriptional regulators. Public Library of Science 2017-09-13 /pmc/articles/PMC5597230/ /pubmed/28902898 http://dx.doi.org/10.1371/journal.pone.0184796 Text en © 2017 Lee et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Lee, Minwoo
Um, Hyejin
Van Dyke, Michael W.
Identification and characterization of preferred DNA-binding sites for the Thermus thermophilus transcriptional regulator FadR
title Identification and characterization of preferred DNA-binding sites for the Thermus thermophilus transcriptional regulator FadR
title_full Identification and characterization of preferred DNA-binding sites for the Thermus thermophilus transcriptional regulator FadR
title_fullStr Identification and characterization of preferred DNA-binding sites for the Thermus thermophilus transcriptional regulator FadR
title_full_unstemmed Identification and characterization of preferred DNA-binding sites for the Thermus thermophilus transcriptional regulator FadR
title_short Identification and characterization of preferred DNA-binding sites for the Thermus thermophilus transcriptional regulator FadR
title_sort identification and characterization of preferred dna-binding sites for the thermus thermophilus transcriptional regulator fadr
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5597230/
https://www.ncbi.nlm.nih.gov/pubmed/28902898
http://dx.doi.org/10.1371/journal.pone.0184796
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