Molecular basis for promoter selectivity of the transcriptional activator OmpR of Escherichia coli: isolation of mutants that can activate the non-cognate kdpABC promoter.
Escherichia coli has two osmo-responsive two-component regulatory systems, the EnvZ-OmpR and KdpD-KdpE systems, each of which consists of a sensor histidine protein kinase and a response regulator. The OmpR and KdpE response regulators belong to the same family of DNA-binding proteins, and act as positive transcriptional factors in response to the medium osmolarity. However, OmpR specifically activates the ompC gene encoding the OmpC outer membrane protein, whereas KdpE exclusively activates the kdpABC operon encoding the high-affinity Kdp potassium-transporter. To gain insight into the molecular basis for such strict promoter selectivity, we isolated OmpR mutants that can activate the non-cognate kdpABC promoter in vivo. For these OmpR mutants, it was found that a few common and crucial amino acids are responsible for the altered property of OmpR (e.g., Gly-164, Glu-193). In vitro properties of these OmpR mutants were further examined by means of DNA-binding assays and DNA-footprinting analyses with reference to the kdpABC promoter. These results were interpreted on the basis of the three-dimensional structure of the C-terminal half of OmpR, which consists of a DNA-binding helix-turn-helix motif and a RNA polymerase-interacting surface. The results of this study were best explained by assuming that the isolated OmpR mutants have an altered property with regard to the interaction with RNA polymerase on the kdpABC promoter. We propose that the promoter selectivity of OmpR is determined not only by its DNA-binding specificity, but also by the spatial configuration of the promoter on which OmpR must properly associate with RNA polymerase.
Ohashi
Kenji
K
Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan.
Yamashino
Takafumi
T
Mizuno
Takeshi
T
D000251:Adenosine Triphosphatases / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D027682:Cation Transport Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D009154:Mutation; D011401:Promoter Regions, Genetic; D015398:Signal Transduction / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli