PMID:9312050
| Citation |
Carmel, O, Rahav-Manor, O, Dover, N, Shaanan, B and Padan, E (1997) The Na+-specific interaction between the LysR-type regulator, NhaR, and the nhaA gene encoding the Na+/H+ antiporter of Escherichia coli. EMBO J. 16:5922-9 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC1170223 Online version:10.1093/emboj/16.19.5922 |
| Keywords |
Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Base Sequence; Binding Sites; DNA Footprinting; DNA Methylation; DNA, Bacterial/chemistry; DNA, Bacterial/metabolism; DNA-Binding Proteins; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Gene Deletion; Histidine/metabolism; Molecular Sequence Data; Promoter Regions, Genetic; Sodium/metabolism; Sodium-Hydrogen Antiporter/genetics; Sodium-Hydrogen Antiporter/metabolism; Transcription Factors/chemistry; Transcription Factors/metabolism |
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