Cyclic AMP receptor protein-dependent activation of the Escherichia coli acsP2 promoter by a synergistic class III mechanism

Abstract

The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by "focusing" RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions -69.5 and -122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the alpha subunit of RNA polymerase holoenzyme (alpha-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the alpha-CTD. Other surface-exposed residues in the alpha-CTD contributed to acs transcription, suggesting that the alpha-CTD may interact with at least one protein other than CRP.

FIG. 1.

Organization of the acs promoter region. (A) The sequence of the acs promoter region from positions −290 to +10 relative to the acsP2 transcription start site (+1). The acsP1, acsP2, and pnrfA −10 elements are underlined, and the predicted start points of transcription for acsP1, acsP2, and pnrfA are designated in bold lowercase letters, with the direction of transcription indicated by a horizontal arrow. Inverted arrows specify the location of DNA sites for CRP binding (CRP I and CRP II), while underlining shows the extent of protection afforded by CRP in DNase I footprint experiments. Triangles indicate hypersensitive sites within the relevant strand. The cleavage sites produced by potassium permanganate footprint analysis are in bold and underlined. (B) Schematic of the region, showing the locations of the +1 sites associated with each promoter, each binding site, and the extent of the pacs444 fragment used for DNase I footprint experiments (Fig. 4).

FIG. 2.

Evidence that P2 is the major acs promoter. (A) Schematic of the acs promoter region, showing the mutant alleles. The sequences of the P1 and P2 −10 elements are underlined. The italic letters above the sequences indicate site-directed mutations. (B) Optical density at 590 nm (OD₅₉₀, dashed lines) and β-galactosidase activity (solid lines) of acs::lacZ transcriptional fusions, either wild type (WT) or mutant for P1 (A214G/A215G) or P2 (C16G/A11C). Wild-type strain AJW678 was lysogenized with a hybrid λ that carried either the wild-type acs::lacZ fusion (λCB12) or one of its mutant derivatives (λCB13 or λCB16). The resultant lysogens were grown in TB, samples were harvested during a growth curve, the β-galactosidase activity was determined, and the activity was expressed as a Miller units. Each value represents the mean ± standard error of the mean (SEM) of at least three independent measurements.

FIG. 3.

CRP binds specifically to two sites within the acs regulatory region. (A) End-labeled pacs444 AatII-HindIII fragment (carrying acs promoter sequences from positions −379 to +65) was incubated with increasing concentrations of CRP protein and subjected to DNase I footprint analysis. The concentration of CRP in each reaction was as follows: lane 1, no protein; lane 2, 30 nM; lane 3, 125 nM; lane 4, 250 nM; lane 5, 500 nM. The gel was calibrated with Maxam-Gilbert G+A sequencing reactions of the labeled fragment (GA). The locations of CRP I and CRP II are shown by boxes, and hypersensitive sites are indicated by stars. The region encompassing the CRP II signature is designated by the thick bar to the right of the figure. (B) A graphic representation of the CRP II region as quantified with ImageQuant (version 5.2) software (Molecular Dynamics). The hypersensitive sites are indicated by stars.

FIG. 4.

CRP activates transcription in vitro by focusing RNAP to P2. (A) CRP is required for transcription of P2 in vitro. In vitro multiround transcription assays of the P2 promoter carried by plasmid pSRacs444. Reactions contained purified RNA polymerase and increasing concentrations of purified CRP. The concentration of CRP was as follows: lane 1, no protein; lane 2, 25 nM; lane 3, 50 nM; lane 4, 100 nM; lane 5, 300 nM. Transcription initiates from P2 and terminates at a strong transcriptional terminator within the vector backbone (pSR), producing a single 139-nucleotide (nt) transcript. The 105-nucleotide RNA I transcript, encoded by pSR, served as an internal transcriptional control. Arrows indicate the P2 and RNA I transcripts. (B) Potassium permanganate footprint analysis of complexes formed at the acs promoter region. The figure shows the cleavage products produced when the end-labeled pacs444 AatII-HindIII fragment was incubated with purified RNA polymerase and purified CRP and then subjected to potassium permanganate footprint analysis. All reactions contained 50 nM RNAP polymerase and the following CRP concentration: lane 2, no protein; lane 3, 25 nM; lane 4, 100 nM. The gel was calibrated with Maxam-Gilbert G+A sequencing reactions of the labeled fragment (GA). The location of the P2 promoter is shown.

FIG. 5.

acs transcription requires CRP I, while optimal transcription also involves CRP II. (A) Schematic of the acs promoter region showing the sequences of CRP I and CRP II. The italic letters and numbers above the sequences indicate site-directed mutations. (B) β-Galactosidase activity of acs::lacZ transcriptional fusions, either wild type or mutant for CRP I (G75C, T76A, G77C, +1A) or CRP II (G126C). Wild-type strain AJW678 was lysogenized with a hybrid λ that carried either the wild-type acs::lacZ fusion (λCB12) or one of its mutant derivatives (λCB15, λCB20, λCB21, λCB21a, or λCB36). The resultant strains were grown in TB, samples were harvested during the transition from exponential growth to stationary phase, the β-galactosidase activity was determined, and the activity was expressed as a percentage of the wild-type value. Each value represents the mean ± SEM of at least three independent measurements. Fold changes in relation to the wild type are noted below the histogram. (C) β-Galactosidase activity of acs::lacZ transcriptional fusions, either wild type (λCB12), mutant (G75C) for CRP I (λCB20), or mutant (G126C) for CRP II (λCB26) carried by cells with crp deleted (strains AJU2163, AJW2181, or AJW2182, respectively) and transformed. The resultant transformants were grown in TB, samples were harvested during the transition from exponential growth to stationary phase, the β-galactosidase activity was determined, and the activity was expressed as a percentage of the wild-type value. Each value represents the mean ± SEM of at least three independent measurements.

FIG. 6.

CRP-dependent activation requires activating region I. β-Galactosidase activity of a λCB12 lysogen deleted for crp (strain AJW2163) and transformed with plasmids expressing either wild-type CRP, CRP H159L, CRP K101E, or CRP H159L/K52N was determined. The resultant transformants were grown in TB, samples were harvested at the transition from exponential to stationary phase, and the β-galactosidase activity was determined and expressed in Miller units. Each value represents the mean ± SEM of at least three independent measurements. Changes in relation to the wild type are noted below the histogram.

FIG. 7.

Effect of single alanine substitutions within α-CTD. The relative β-galactosidase activity of a λCB12 lysogen wild type for crp (AJW1941) and transformed with a set of plasmids expressing a library of alanine-substituted variants of the α-CTD of RNAP (residues 255 to 329) was determined. Residues 267, 272, 274, 308, 324, and 327 are naturally alanines. The resultant transformants were grown in TB, samples were harvested during the transition from exponential growth to stationary phase, and the β-galactosidase activity was determined. Activities are expressed as a percentage of that of the transformant containing a plasmid expressing wild-type α. Each value represents the mean ± SEM of at least two independent measurements. All alanine substitutions that significantly altered expression of the acs::lacZ fusion are marked with an asterisk above the bar. Horizontally striped bars, the 261 determinant; open bars, the 265 determinant; diagonally striped bars, the 287 determinant; cross-hatched bars, residues outside a known determinant.