Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.

DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The "genetic switch" of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Zach Hensel, Xiaoli Weng, Arvin Cesar Lagda, Jie Xiao
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/260c41e554f34ee1a1db701629ce7e6c
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:260c41e554f34ee1a1db701629ce7e6c
record_format	dspace
spelling	oai:doaj.org-article:260c41e554f34ee1a1db701629ce7e6c2021-11-18T05:37:02ZTranscription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.1544-91731545-788510.1371/journal.pbio.1001591https://doaj.org/article/260c41e554f34ee1a1db701629ce7e6c2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23853547/pdf/?tool=EBIhttps://doaj.org/toc/1544-9173https://doaj.org/toc/1545-7885DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The "genetic switch" of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences O(R) and O(L) (separated by 2.3 kb), mediated by the λ repressor CI (accession number P03034), play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms.Zach HenselXiaoli WengArvin Cesar LagdaJie XiaoPublic Library of Science (PLoS)articleBiology (General)QH301-705.5ENPLoS Biology, Vol 11, Iss 6, p e1001591 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Biology (General) QH301-705.5
spellingShingle	Biology (General) QH301-705.5 Zach Hensel Xiaoli Weng Arvin Cesar Lagda Jie Xiao Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.
description	DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The "genetic switch" of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences O(R) and O(L) (separated by 2.3 kb), mediated by the λ repressor CI (accession number P03034), play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms.
format	article
author	Zach Hensel Xiaoli Weng Arvin Cesar Lagda Jie Xiao
author_facet	Zach Hensel Xiaoli Weng Arvin Cesar Lagda Jie Xiao
author_sort	Zach Hensel
title	Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.
title_short	Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.
title_full	Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.
title_fullStr	Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.
title_full_unstemmed	Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.
title_sort	transcription-factor-mediated dna looping probed by high-resolution, single-molecule imaging in live e. coli cells.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/260c41e554f34ee1a1db701629ce7e6c
work_keys_str_mv	AT zachhensel transcriptionfactormediateddnaloopingprobedbyhighresolutionsinglemoleculeimaginginliveecolicells AT xiaoliweng transcriptionfactormediateddnaloopingprobedbyhighresolutionsinglemoleculeimaginginliveecolicells AT arvincesarlagda transcriptionfactormediateddnaloopingprobedbyhighresolutionsinglemoleculeimaginginliveecolicells AT jiexiao transcriptionfactormediateddnaloopingprobedbyhighresolutionsinglemoleculeimaginginliveecolicells
_version_	1718424838333267968

Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.

Ejemplares similares