| The Nar two-component system of Escherichia coli responds to nitrate and nitrite under the anaerobic condition by transcription activation or repression of the genes needed for anaerobic respiration and fermentation. The system consists of two membrane-bound sensor/transmitters, NarX and NarQ, and two cytoplasmic response regulators, NarL and NarP. NarL as the intermediate of the signal transduction pathway is regulated by phosphorylation and dephosphorylation by sensorltransmitters, and regulates the transcription of the downstream genes by DNA-binding and/or interaction with other transcription factors andlor RNA polymerase. NarL consists of two domains, the N-terminal phosphorylation domain (NarLN) and the C-terminal DNA-binding domain (NarL C), tethered by a linker region.; NarL was covalently modified with 1,10-phenanthroline to create two classes of site-specific DNA cleaving agents. The first class cleaved DNA nearby the center of a synthetic 7-2-7 sequence composed of two NarL heptamers separated by a two-basepair spacer. The second class cut DNA at the periphery of the 7-2-7 sequence. The cleavage data demonstrate that two NarL monomers recognize and bind to the DNA in a head to head orientation. Similar cleavage patterns were observed for full length NarL and the isolated NarLC.; The isolated unphosphorylated NarLN formed a complex with the isolated NarLC. The linker region is not required for the interdomain interaction under the unphosphorylation status. Phosphorylation triggered the separation of the two domains, the exposure of the residue 204 in NarLC, and the distance increase between the residue 78 in NarLN and the residue 196 in NarLC.; The alanine scanning mutagenesis strategy was used to extensively screen residues in NarLN important for phosphorylation, dephosphorylation, and protein-protein interaction in transcription activation. At least three classes of NarL mutants deficient in one of its functions were obtained. Preliminary data suggested that the phosphorylation and dephosphorylation surfaces of NarL are mostly distinct. |
