Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K-12 depends on DNA binding site arrangement

The NarL and NarP proteins are homologous response regulators of Escherichia coli that control the expression of several operons in response to nitrate and nitrite. A consensus heptameric NarL DNA-binding sequence has been identified, and previous observations suggest that the NarP protein has a similar sequence specificity. However, some operons are regulated by NarL alone, whereas others are controlled by both NarL and Harp. In this study, DNase I footprinting experiments with the fdnG, nirB and nrfA control regions revealed that NarP only binds to heptamer sequences organized as an inverted repeat with a 2 bp spacing (7-2-7 sites). The NarL protein also binds to these 7-2-7 sites but, unlike Harp, also recognizes heptamers in other arrangements. These results provide an explanation for the regulation of some operons by NarL alone and for the different effects of NarL and Harp at common target operons, such as fdnG and nrfA. To investigate this differential DNA binding further, derivatives of the nrfA control region were constructed in which the spacing of the 7-2-7 heptamers was increased (7-n-7 constructs). Increasing the spacing to four or more basepairs abolished Harp binding and significantly reduced NarL binding. The NarL protein also had a reduced binding affinity for heptamers adjacent to the 7-n-7 heptamer pair, suggesting a decrease in cooperative interactions. In conclusion, we propose that 7-2-7 sites are preferred by both NarL and NarP. NarL can also recognize other binding site arrangements, an ability that appears to be lacking in NarP.