The Na+-specific interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na+/H+ antiporter of Escherichia coli
Autor: | Nir Dover, O. Rahav-Manor, Etana Padan, O. Carmel, B. Shaanan |
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Rok vydání: | 1997 |
Předmět: |
DNA
Bacterial Conformational change Sodium-Hydrogen Exchangers Molecular Sequence Data DNA Footprinting DNA footprinting Biology medicine.disease_cause General Biochemistry Genetics and Molecular Biology Bacterial Proteins Escherichia coli medicine Histidine Electrophoretic mobility shift assay Amino Acid Sequence Binding site Promoter Regions Genetic Molecular Biology Binding Sites Base Sequence General Immunology and Microbiology Escherichia coli Proteins General Neuroscience Sodium Methylation DNA Methylation Molecular biology DNA-Binding Proteins Sodium–hydrogen antiporter Gene Deletion Research Article Transcription Factors |
Zdroj: | The EMBO Journal. 16:5922-5929 |
ISSN: | 1460-2075 0261-4189 |
Popis: | We used partially purified NhaR and a highly purified His-tagged NhaR derivative to identify the cis-regulatory sequences of nhaA recognized by NhaR and to study the specific effect of Na+ on this interaction. Gel retardation assay with DNase I footprinting analysis showed that NhaR binds a region of nhaA which spans 92 bp and contains three copies of the conserved LysR-binding motif. Na+, up to 100 mM, had no effect on the binding of NhaR to nhaA. The dimethylsulfate methylation protection assay in vivo and in vitro, showed that bases G-92, G-60, G-29 and A-24 form direct contacts with NhaR; in the absence of added Na+ in vivo, these bases were protected but became exposed to methylation in a DeltanhaR strain; accordingly, these bases were protected in vitro by the purified His-tagged NhaR. 100 mM Na+, but not K+, removed the protection of G-60 conferred by His-tagged NhaR in vitro. Exposure of intact cells to 100 mM Na+, but not K+, exposed G-60. The maximal effect of Na+ in vitro was observed at 20 mM and was pH dependent, vanishing below pH 7.5. In contrast to G-60, G-92 was exposed to methylation by the ion only in vivo, suggesting a requirement for another factor existing only in vivo for this interaction. We suggest that NhaR is both sensor and transducer of the Na+ signal and that it regulates nhaA expression by undergoing a conformational change upon Na+ binding which modifies the NhaR-nhaA contact points. |
Databáze: | OpenAIRE |
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