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

Abstract

SummaryThe 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 NarP. 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 NarP, 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 NarP 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 NarP 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.

Sensor–response regulator interactions in a cross-regulated signal transduction network

“…Receiver phosphorylation by the sensors results in effector domain dimerization, enhancing affinity for DNA binding Maris et al, 2002). The two proteins recognize dissimilar DNA-binding site architectures (Darwin et al, 1997), contributing to differential target operon control.…”

Characterization of the Escherichia coli K-12 ydhYVWXUT operon: regulation by FNR, NarL and NarP

“…These sensors then act to control the phosphorylation state of the regulators NarL and NarP (Darwin & Stewart, 1996;Yamamoto et al, 2005), and there is a complex exchange of information between the sensors and the regulators to allow fine control of targetgene expression in response to nitrate and nitrite. The phosphorylated regulators bind specific DNA sequences related to the consensus TACYYMT (where Y5C or T, and M5A or C) to positively or negatively regulate gene expression (Darwin et al, 1997). In addition, there is differential recognition of NarL/P heptamer patterning, such that NarL recognizes all TACYYMT heptamers, whereas NarP only binds these sequences when they are arranged as an inverted repeat with a 2 bp spacing, the socalled 7-2-7 arrangement (Darwin et al, 1997).…”

“…The phosphorylated regulators bind specific DNA sequences related to the consensus TACYYMT (where Y5C or T, and M5A or C) to positively or negatively regulate gene expression (Darwin et al, 1997). In addition, there is differential recognition of NarL/P heptamer patterning, such that NarL recognizes all TACYYMT heptamers, whereas NarP only binds these sequences when they are arranged as an inverted repeat with a 2 bp spacing, the socalled 7-2-7 arrangement (Darwin et al, 1997). Thus, the Nar two-component systems work in conjunction with FNR to maintain a central metabolic hierarchy in E. coli.…”

“…After electrophoresis the gels were transferred to filter paper (3MM, Whatman) and dried for autoradiography. Footprinting reactions with FNR* (2 mM), phosphorylated MBPNarL (0-6 mM) and phosphorylated MBP-NarP (0-6 mM) were done essentially as described by Darwin et al (1997) with PydhY (2189 to +176) used to construct pGS1739 as the target DNA (see above). The promoter fragment was radiolabelled using Klenow fragment and an appropriate a[ 32 P]dNTP at either the BamHI site (NarP) or the EcoRI site (NarL).…”

“…The promoter fragment was radiolabelled using Klenow fragment and an appropriate a[ 32 P]dNTP at either the BamHI site (NarP) or the EcoRI site (NarL). The MBP-NarL and MBP-NarP proteins were purified as previously described (Darwin et al, 1997).…”