Binding of PhoP to promoters of phosphate‐regulated genes in Streptomyces coelicolor: identification of PHO boxes

[EN] The control of phosphate-regulated genes in Streptomyces coelicolor is mediated by the two-component system PhoR–PhoP. When coupled to the reporter xylE gene the pstS, phoRP and phoU promoters were shown to be very sensitive to phosphate regulation. The transcription start points of the pstS, t...

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Autores: Sola Landa, Alberto, Rodríguez García, Antonio, Franco Domínguez, Etelvina, Martín Martín, Juan Francisco
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2005
País:España
Institución:Universidad Rey Juan Carlos
Repositorio:BULERIA. Repositorio Institucional de la Universidad de León
OAI Identifier:oai:buleria.unileon.es:10612/17742
Acceso en línea:https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2005.04631.x
https://hdl.handle.net/10612/17742
Access Level:acceso abierto
Palabra clave:Biotecnología
Streptomyces
Phosphate regulation
PHO boxes
PhoR-PhoP
PhoP binding sites
DNAse footprinting
Primer extension
EMSA
Binding assay
Protein purification
2415.01 Biología Molecular de Microorganismos
id ES_2bca06df0859ef54cfc29c19e5fcda05
oai_identifier_str oai:buleria.unileon.es:10612/17742
network_acronym_str ES
network_name_str España
spelling Binding of PhoP to promoters of phosphate‐regulated genes in Streptomyces coelicolor: identification of PHO boxes Sola Landa, Alberto Rodríguez García, Antonio Franco Domínguez, Etelvina Martín Martín, Juan Francisco Biotecnología Streptomyces Phosphate regulation PHO boxes PhoR-PhoP PhoP binding sites DNAse footprinting Primer extension EMSA Binding assay Protein purification 2415.01 Biología Molecular de Microorganismos [EN] The control of phosphate-regulated genes in Streptomyces coelicolor is mediated by the two-component system PhoR–PhoP. When coupled to the reporter xylE gene the pstS, phoRP and phoU promoters were shown to be very sensitive to phosphate regulation. The transcription start points of the pstS, the phoRP and the phoU promoters were identified by primer extension. phoRP showed a leaderless transcript. The response-regulator (DNA-binding) PhoP protein was overexpressed and purified in Escherichia coli as a GST–PhoP fused protein. The DNA-binding domain (DBD) of PhoP was also obtained in a similar manner. Both PhoP and its truncated DBD domain were found to bind with high affinity to an upstream region of the pstS and phoRP–phoU promoters close to the −35 sequence of each of these promoters. DNase I protection studies revealed a 29 bp protected stretch in the sense strand of the pstS promoter that includes two 11 bp direct repeat units. Footprinting of the bidirectional phoRP–phoU promoter region showed a 51 bp protected sequence that encompasses four direct repeat units, two of them with high similarity to the protected sequences in the pstS promoter. PHO boxes have been identified by alignment of the six direct repeat units found in those promoter regions. Each direct repeat unit adjusts to the consensus GG/TTCAYYYRG/CG SI This work was supported by Grants BIO2003-01489 and GEN2003-20245-C09-01-NAC of the CICYT, Ministry of Science and Technology, Madrid, Spain. E.F.-D. received a fellowship of the Diputación de León, Spain. We thank J. Merino, B. Martín, A. Casenave and M. Álvarez for the excellent technical assistance. BLACKWELL PUBLISHING https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2005.04631.x https://hdl.handle.net/10612/17742
title Binding of PhoP to promoters of phosphate‐regulated genes in Streptomyces coelicolor: identification of PHO boxes
spellingShingle Binding of PhoP to promoters of phosphate‐regulated genes in Streptomyces coelicolor: identification of PHO boxes
Sola Landa, Alberto
Biotecnología
Streptomyces
Phosphate regulation
PHO boxes
PhoR-PhoP
PhoP binding sites
DNAse footprinting
Primer extension
EMSA
Binding assay
Protein purification
2415.01 Biología Molecular de Microorganismos
title_short Binding of PhoP to promoters of phosphate‐regulated genes in Streptomyces coelicolor: identification of PHO boxes
title_full Binding of PhoP to promoters of phosphate‐regulated genes in Streptomyces coelicolor: identification of PHO boxes
title_fullStr Binding of PhoP to promoters of phosphate‐regulated genes in Streptomyces coelicolor: identification of PHO boxes
title_full_unstemmed Binding of PhoP to promoters of phosphate‐regulated genes in Streptomyces coelicolor: identification of PHO boxes
title_sort Binding of PhoP to promoters of phosphate‐regulated genes in Streptomyces coelicolor: identification of PHO boxes
author Sola Landa, Alberto
author_facet Sola Landa, Alberto
Rodríguez García, Antonio
Franco Domínguez, Etelvina
Martín Martín, Juan Francisco
author_role author
author2 Rodríguez García, Antonio
Franco Domínguez, Etelvina
Martín Martín, Juan Francisco
author2_role author
author
author
topic Biotecnología
Streptomyces
Phosphate regulation
PHO boxes
PhoR-PhoP
PhoP binding sites
DNAse footprinting
Primer extension
EMSA
Binding assay
Protein purification
2415.01 Biología Molecular de Microorganismos
topic_facet Biotecnología
Streptomyces
Phosphate regulation
PHO boxes
PhoR-PhoP
PhoP binding sites
DNAse footprinting
Primer extension
EMSA
Binding assay
Protein purification
2415.01 Biología Molecular de Microorganismos
description [EN] The control of phosphate-regulated genes in Streptomyces coelicolor is mediated by the two-component system PhoR–PhoP. When coupled to the reporter xylE gene the pstS, phoRP and phoU promoters were shown to be very sensitive to phosphate regulation. The transcription start points of the pstS, the phoRP and the phoU promoters were identified by primer extension. phoRP showed a leaderless transcript. The response-regulator (DNA-binding) PhoP protein was overexpressed and purified in Escherichia coli as a GST–PhoP fused protein. The DNA-binding domain (DBD) of PhoP was also obtained in a similar manner. Both PhoP and its truncated DBD domain were found to bind with high affinity to an upstream region of the pstS and phoRP–phoU promoters close to the −35 sequence of each of these promoters. DNase I protection studies revealed a 29 bp protected stretch in the sense strand of the pstS promoter that includes two 11 bp direct repeat units. Footprinting of the bidirectional phoRP–phoU promoter region showed a 51 bp protected sequence that encompasses four direct repeat units, two of them with high similarity to the protected sequences in the pstS promoter. PHO boxes have been identified by alignment of the six direct repeat units found in those promoter regions. Each direct repeat unit adjusts to the consensus GG/TTCAYYYRG/CG
publishDate 2005
format article
status_str publishedVersion
url https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2005.04631.x
https://hdl.handle.net/10612/17742
eu_rights_str_mv openAccess
publisher BLACKWELL PUBLISHING
institution Universidad Rey Juan Carlos
collection BULERIA. Repositorio Institucional de la Universidad de León
reponame_str BULERIA. Repositorio Institucional de la Universidad de León
instname_str Universidad Rey Juan Carlos
_version_ 1878432847085174784
publishDateSort 2005
author_browse Franco Domínguez, Etelvina
Martín Martín, Juan Francisco
Rodríguez García, Antonio
Sola Landa, Alberto
publisherStr BLACKWELL PUBLISHING
score 6,924472

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