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Journal of Bacteriology, July 2001, p. 3910-3918, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.3910-3918.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Activation from a Distance: Roles of Lrp and Integration Host Factor in Transcriptional Activation of gltBDF

Ligi Paul,1 Robert M. Blumenthal,2 and Rowena G. Matthews1,3,*

Biophysics Research Division1 and Department of Biological Chemistry,3 University of Michigan, Ann Arbor, Michigan 48109, and Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 436142

Received 23 January 2001/Accepted 13 April 2001

The leucine-responsive regulatory protein (Lrp) binds to three sites centered 252, 216, and 152 bp upstream of the transcription start site of the Escherichia coli glutamate synthase operon (gltBDF) and activates transcription. Activators of sigma 70-dependent promoters usually bind closer to the -35 hexamer of the core promoter sequence. To study the mechanism by which Lrp-dependent activation occurs over this relatively large distance, the gltBDF upstream region was sequentially replaced with corresponding portions from the well-characterized sigma 70-dependent promoter lacZYAp. The glt-lac promoter hybrids were placed upstream of lacZ, allowing transcriptional activity to be monitored via beta -galactosidase assays. Even replacing all gltBDF sequences downstream of and including the -35 hexamer did not eliminate Lrp-dependent activation of transcription. When a 91-bp region between the -35 hexamer and the proximal Lrp binding site (-48 to -128) was replaced with heterologous DNA of the same length, transcription was reduced nearly 40-fold. Based on the presence of a consensus binding sequence, this region seemed likely to be a binding site for integration host factor (IHF). Experiments to study the effects of a himD mutant on expression of a gltB::lacZ transcriptional fusion, gel mobility shift analyses, and DNA footprinting assays were used to confirm the direct participation of IHF in gltBDF promoter regulation. Based on these results, we suggest that IHF plays a crucial architectural role, bringing the distant Lrp complex in close proximity to the promoter-bound RNA polymerase.

* Corresponding author. Mailing address: Biophysics Research Division, 4028 Chemistry, 930 N. University Ave, University of Michigan, Ann Arbor, MI 48109-1055. Phone: (734) 764-5257. Fax: (734) 764-3323. E-mail rmatthew{at}umich.edu.

Journal of Bacteriology, July 2001, p. 3910-3918, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.3910-3918.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.