| Escherichia coli NarL and NarP are response regulators that mediate anaerobic gene expression under nitrate regulation. NarL and NarP are composed of N-terminal domains (NTD) that are phosphorylated by NarX and NarQ sensor-histidine kinases and C-terminal domains (CTD) that bind to 7-2-7 heptamer pairs. The X-ray structure of unphosphorylated monomeric NarL protein shows the N-terminal receiver domain positioned to block the carboxyl-terminal DNA binding domain, suggesting that DNA binding requires intramolecular domain rearrangement. The biochemical and X-ray studies of the CTD show that it contains all determinants necessary for specific DNA binding. In this study, we tested NarL-CTD and NarP-CTD in transcription activation, transcription repression, and in vitro DNA binding. We found NarL-CTD and NarP-CTD can function for transcription activation in one specific context, the Fnr-dependent Nar class II control regions. However, in all other cases examined, including repression at the synthetic lac operators, NarL-NTD and NarP-NTD are required for full transcriptional function. We further performed selective alanine scanning of NarL-CTD based on its homology with TraR-CTD. Each mutant was tested for transcription activation and repression in vivo. NarL R178A and R179A had wild-type level repression in vivo and in vitro but are defective in transcription at Fnr-dependent class II promoters. We propose that these two residues make direct contact with the RNA polymerase at Fnr-dependent class II promoters. |
