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Journal of Bacteriology, December 1999, p. 7266-7273, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

RNA Polymerase alpha  and sigma 70 Subunits Participate in Transcription of the Escherichia coli uhpT Promoter

Igor N. Olekhnovich and Robert J. Kadner*

Department of Microbiology, School of Medicine, University of Virginia, Charlottesville, Virginia 22908

Received 6 July 1999/Accepted 17 September 1999

Fundamental questions in bacterial gene regulation concern how multiple regulatory proteins interact with the transcription apparatus at a single promoter and what are the roles of protein contacts with RNA polymerase and changes in DNA conformation. Transcription of the Escherichia coli uhpT gene, encoding the inducible sugar phosphate transporter, is dependent on the response regulator UhpA and is stimulated by the cyclic AMP receptor protein (CAP). UhpA binds to multiple sites in the uhpT promoter between positions -80 and -32 upstream of the transcription start site, and CAP binds to a single site centered at position -103.5. The role in uhpT transcription of portions of RNA polymerase Esigma 70 holoenzyme which affect regulation at other promoters was examined by using series of alanine substitutions throughout the C-terminal domains of RpoA (residues 255 to 329) and of RpoD (residues 570 to 613). Alanine substitutions that affected in vivo expression of a uhpT-lacZ transcriptional fusion were tested for their effect on in vitro transcription activity by using reconstituted holoenzymes. Consistent with the binding of UhpA near the -35 region, residues K593 and K599 in the C-terminal region of RpoD were necessary for efficient uhpT expression in response to UhpA alone. Their requirement was overcome when CAP was also present. In addition, residues R265, G296, and S299 in the DNA-binding surface of the C-terminal domain of RpoA (alpha CTD) were important for uhpT transcription even in the presence of CAP. Substitutions at several other positions had effects in cells but not during in vitro transcription with saturating levels of the transcription factors. Two DNase-hypersensitive sites near the upstream end of the UhpA-binding region were seen in the presence of all three transcription factors. Their appearance required functional alpha CTD but not the presence of upstream DNA. These results suggest that both transcription activators depend on or interact with different subunits of RNA polymerase, although their role in formation of proper DNA geometry may also be crucial.

* Corresponding author. Mailing address: Department of Microbiology HSC#441, University of Virginia, Charlottesville, VA 22908. Phone: (804) 924 2532. Fax: (804) 982 1071. E-mail: rjk{at}virginia.edu.

Journal of Bacteriology, December 1999, p. 7266-7273, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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