microbiology, Vol 143, 1521-1532, Copyright © 1997 by Society for General Microbiology

ARTICLES

FNR-dependent repression of ndh gene expression requires two upstream FNR-binding sites

W Meng, J Green and JR Guest
Krebs Institute for Biomolecular Research, Department of Molecular Biology & Biotechnology, University of Sheffield, Western Bank, UK.

The ndh gene of Escherichia coli encodes a non-proton-translocating NADH dehydrogenase (NdhII) that is anaerobically repressed by the global transcription regulator, FNR. FNR binds at two sites (centred at -50.5 and -94.5) in the ndh promoter but the mechanism of FNR-mediated repression appears not to be due to promoter occlusion. This mechanism has been investigated using an aerobically active derivative of FNR, FNR* (FNR-D154A), with ndh promoters containing altered FNR-binding sites. FNR* repressed ndh gene expression both aerobically and anaerobically in vivo. Gel retardation analysis and DNase I footprinting with purified FNR* protein confirmed that FNR interacts at two sites in the ndh promoter, and that FNR and RNA polymerase (RNAP) can bind simultaneously. Studies with three altered ndh promoters, each containing an impaired or improved FNR-site, indicated that both FNR- sites are needed for efficient repression in vivo. The alpha-subunit of RNAP interacted with two regions (centred at -105 and -46), each overlapping one of the FNR-sites in the ndh promoter. Footprints of the FNR*-RNAP-ndh ternary complex indicated that FNR*-binding at -50.5 prevents the alpha-subunit of RNAP from docking with the DNA just upstream of the -35 element. Binding of a second FNR* molecule at the - 105 site likewise prevents binding of the alpha-subunit at its alternative site, thus providing a plausible mechanism for FNR-mediated repression based on displacement of the alpha-subunit of RNAP.

This article has been cited by other articles:

				H. Ogasawara, Y. Ishida, K. Yamada, K. Yamamoto, and A. Ishihama PdhR (Pyruvate Dehydrogenase Complex Regulator) Controls the Respiratory Electron Transport System in Escherichia coli J. Bacteriol., August 1, 2007; 189(15): 5534 - 5541. [Abstract] [Full Text] [PDF]

				D. P. Dibden and J. Green In vivo cycling of the Escherichia coli transcription factor FNR between active and inactive states Microbiology, December 1, 2005; 151(12): 4063 - 4070. [Abstract] [Full Text] [PDF]

				R. G. Sawers Expression of fnr Is Constrained by an Upstream IS5 Insertion in Certain Escherichia coli K-12 Strains J. Bacteriol., April 15, 2005; 187(8): 2609 - 2617. [Abstract] [Full Text] [PDF]

				Y. Kang, K. D. Weber, Y. Qiu, P. J. Kiley, and F. R. Blattner Genome-Wide Expression Analysis Indicates that FNR of Escherichia coli K-12 Regulates a Large Number of Genes of Unknown Function J. Bacteriol., February 1, 2005; 187(3): 1135 - 1160. [Abstract] [Full Text] [PDF]

				V. R. Sutton, E. L. Mettert, H. Beinert, and P. J. Kiley Kinetic Analysis of the Oxidative Conversion of the [4Fe-4S]2+ Cluster of FNR to a [2Fe-2S]2+ Cluster J. Bacteriol., December 1, 2004; 186(23): 8018 - 8025. [Abstract] [Full Text] [PDF]

				L. Jackson, T. Blake, and J. Green Regulation of ndh expression in Escherichia coli by Fis Microbiology, February 1, 2004; 150(2): 407 - 413. [Abstract] [Full Text] [PDF]

				A. M. L. Barnard, J. Green, and S. J. W. Busby Transcription Regulation by Tandem-Bound FNR at Escherichia coli Promoters J. Bacteriol., October 15, 2003; 185(20): 5993 - 6004. [Abstract] [Full Text] [PDF]

				K. Salmon, S.-p. Hung, K. Mekjian, P. Baldi, G. W. Hatfield, and R. P. Gunsalus Global Gene Expression Profiling in Escherichia coli K12: THE EFFECTS OF OXYGEN AVAILABILITY AND FNR J. Biol. Chem., August 8, 2003; 278(32): 29837 - 29855. [Abstract] [Full Text] [PDF]

				M. Marino, T. Hoffmann, R. Schmid, H. Möbitz, and D. Jahn Changes in protein synthesis during the adaptation of Bacillus subtilis to anaerobic growth conditions Microbiology, January 1, 2000; 146(1): 97 - 105. [Abstract] [Full Text]

				J. Green and F. A. Marshall Identification of a Surface of FNR Overlapping Activating Region 1 That Is Required for Repression of Gene Expression J. Biol. Chem., April 9, 1999; 274(15): 10244 - 10248. [Abstract] [Full Text] [PDF]

				M. K. B. Berlyn Linkage Map of Escherichia coli K-12, Edition 10: The Traditional Map Microbiol. Mol. Biol. Rev., September 1, 1998; 62(3): 814 - 984. [Abstract] [Full Text] [PDF]

				C. Scott and J. Green Miscoordination of the Iron-Sulfur Clusters of the Anaerobic Transcription Factor, FNR, Allows Simple Repression but Not Activation J. Biol. Chem., January 11, 2002; 277(3): 1749 - 1754. [Abstract] [Full Text] [PDF]