Factors influencing transcription activation by response regulators NarP and NarL of Escherichia coli.

摘要

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.