RNA polymerase alpha and sigma(70) subunits participate in transcription of the Escherichia coli uhpT promoter

Abstract

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 (alphaCTD) 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 alphaCTD 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.

FIG. 1

Effect of alanine substitutions in ςCTD on uhpT-lacZ expression in vivo. (A) Results with host strain RK1309; (B) results with host strain IO607. Values of β-galactosidase activity are averages from three independent experiments and are presented as percentages of the wild-type (WT) values (270 and 9 Miller units, respectively). Residues 609 to 611 are alanines. Error bars indicate standard deviations.

FIG. 2

Changes in uhpT-lacZ expression in response to the presence of CAP and variations in RpoA and RpoD. Host strains were crp⁺ (A and C) and Δcrp (B and D). (A and B) Plasmids encoding wild-type RpoD (open bars), RpoD-E570A (shaded bars), and RpoD-D575A (cross-hatched bars). (C and D) Plasmids encoding wild-type RpoA (open bars) and RpoA-Δ256 (filled bars). Values are β-galactosidase activities in Miller units. Error bars indicate standard deviations.

FIG. 3

Transcription of the uhpT and lacUV5 promoters by wild-type (WT) RNAP and mutant RNAPs carrying RpoD variants E570A, D575A, E591A, K593A, L598A, and R599A. Reactions were carried out in the absence and presence of 20 nM CAP, as indicated. (A) The ratios of the amounts of the uhpT and the lac transcripts were determined by PhosphorImager quantitation. Transcription of uhpT was measured in the presence of UhpA. (B) uhpT transcription (Txn) in the presence of UhpA without and with CAP as indicated, calculated relative to the amount of transcription produced by the wild-type RNAP in the presence of UhpA.

FIG. 4

Effect of alanine substitutions in αCTD on uhpT-lacZ expression in vivo. Values of β-galactosidase activity are averages from three independent experiments and are plotted as percentages of the wild-type (WT) value (370 Miller units). Error bars indicate standard deviations.

FIG. 5

Transcription of the uhpT and lacUV5 promoters by wild-type (WT) RNAP and mutant RNAPs carrying the indicated alanine substitutions in RpoA. The relative amounts of the uhpT transcript (Txn level), determined by PhosphorImager quantitation, are presented relative to that for the wild-type RNAP in the presence of UhpA.

FIG. 6

DNase I footprinting at the uhpT promoter region. (A) UhpA, CAP, and RNAP variants were present as indicated on the top. The positions protected by UhpA and CAP are indicated on the left, and the positions of the DNase hypersensitive sites are indicated on the right. RNAP samples contained a 5 nM concentration of the wild-type (WT), 265A, or 299A form of RpoA, as indicated. (B) Three DNA fragments whose upstream ends were at positions −121, −179, and −250, as indicated, were used. For each DNA fragment, the sample in lane 1 contained DNA, that in lane 2 contained DNA, 220 nM UhpA, and 80 nM CAP, and that in lane 3 contained DNA, UhpA, CAP, and 10 nM WT RNAP.