title: Three of four GlnR binding sites are essential for GlnR-mediated activation of transcription of the Amycolatopsis mediterranei nas operon.
abstract: In Amycolatopsis mediterranei U32, genes responsible for nitrate assimilation formed one operon, nasACKBDEF, whose transcription is induced by the addition of nitrate. Here, we characterized GlnR as a direct transcriptional activator for the nas operon. The GlnR-protected DNA sequences in the promoter region of the nas operon were characterized by DNase I footprinting assay, the previously deduced Streptomyces coelicolor double 22-bp GlnR binding consensus sequences comprising a1, b1, a2, and b2 sites were identified, and the sites were then mutated individually to test their roles in both the binding of GlnR in vitro and the GlnR-mediated transcriptional activation in vivo. The results clearly showed that only three GlnR binding sites (a1, b1, and b2 sites) were required by GlnR for its specific binding to the nas promoter region and efficient activation of the transcription of the nas operon in U32, while the a2 site seemed unnecessary.
authors.affiliation: CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Jing-Zhi
authors.initials: JZ
authors.lastname: Wang
authors.forename: Zhi-Hui
authors.initials: ZH
authors.lastname: Shao
authors.forename: Hua
authors.initials: H
authors.lastname: Yuan
authors.forename: Yin-Hua
authors.initials: YH
authors.lastname: Lu
authors.forename: Wei-Hong
authors.initials: WH
authors.lastname: Jiang
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Zhao
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
mesh_terms: D000192:Actinomycetales / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D011401:Promoter Regions, Genetic / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D015534:Trans-Activators
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