Darwin, Andrew J. and Ziegelhoffer, Eva C. and Kiley, Patricia J. and Stewart, Valley (1998) Fnr, NarP, and NarL Regulation of Escherichia coli K-12 napF (Periplasmic Nitrate Reductase) Operon Transcription In Vitro. Journal of Bacteriology, 180 (16). pp. 4192-4198. ISSN 0021-9193. http://resolver.caltech.edu/CaltechAUTHORS:DARjbact98
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The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.
|Additional Information:||Copyright © 1998, American Society for Microbiology. Received 16 March 1998/Accepted 17 June 1998 A.D. thanks the members of the Kiley laboratory for their help and hospitality during his visit, during which some of these experiments were initiated. We thank Steve Busby for the gift of plasmids and for many helpful discussions. This study was supported by Public Health Service grants GM36877 (awarded to V.S., Cornell University) and GM45844 (awarded to P.J.K., University of Wisconsin) from the National Institute of General Medical Sciences; a National Science Foundation Young Investigator Award (awarded to P.J.K.); and predoctoral National Research Service Award GM07215 (awarded to E.C.Z., University of Wisconsin) from the NIGMS.|
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