ExsA recruits RNA polymerase to an extended -10 promoter by contacting region 4.2 of sigma-70

Abstract

ExsA is a member of the AraC family of transcriptional activators and is required for expression of the Pseudomonas aeruginosa type III secretion system (T3SS). ExsA-dependent promoters consist of two binding sites for monomeric ExsA located approximately 50 bp upstream of the transcription start sites. Binding to both sites is required for recruitment of sigma(70)-RNA polymerase (RNAP) to the promoter. ExsA-dependent promoters also contain putative -35 hexamers that closely match the sigma(70) consensus but are atypically spaced 21 or 22 bp from the -10 hexamer. Because several nucleotides located within the putative -35 region are required for ExsA binding, it is unclear whether the putative -35 region makes an additional contribution to transcription initiation. In the present study we demonstrate that the putative -35 hexamer is dispensable for ExsA-independent transcription from the P(exsC) promoter and that deletion of sigma(70) region 4.2, which contacts the -35 hexamer, has no effect on ExsA-independent transcription from P(exsC). Region 4.2 of sigma(70), however, is required for ExsA-dependent activation of the P(exsC) and P(exsD) promoters. Genetic data suggest that ExsA directly contacts region 4.2 of sigma(70), and several amino acids were found to contribute to the interaction. In vitro transcription assays demonstrate that an extended -10 element located in the P(exsC) promoter is important for overall promoter activity. Our collective data suggest a model in which ExsA compensates for the lack of a -35 hexamer by interacting with region 4.2 of sigma(70) to recruit RNAP to the promoter.

FIG. 1.

The RNAP α-CTD is not required for ExsA-dependent activation of transcription. (A) E. coli strain GS162 carrying the indicated transcriptional reporters (P_exsC-lacZ, P_exsD-lacZ, or P_exoT-lacZ) was transformed with a vector control (pJN105) or a constitutive ExsA expression plasmid (p2UY21, labeled pExsA in the figure). The resulting strains were grown in LB to an OD₆₀₀ of 0.6 and assayed for β-galactosidase activity (reported in Miller units). (B) E. coli GS162 carrying a P_luxI-lacZ reporter and a LuxR expression plasmid (p2UY21-luxR) and the reporter strains from panel A were transformed with a plasmid expressing the native α or αΔCTD subunit. The resulting strains were grown in LB to an OD₆₀₀ of 0.6 and assayed for β-galactosidase activity. The reporter activities obtained in cells expressing αΔCTD were normalized to the same strain expressing native (WT) α and reported as the percentage of native activity. The results represent the averages for three independent experiments, and error bars represent the standard errors of the means.

FIG. 2.

ExsA-dependent transcription requires several amino acids in region 4.2 of E. coli σ⁷⁰. (A) ExsA immunoblots demonstrating that steady-state expression levels are similar in each of the strains used below. (B and C) E. coli strain GA2071 (tightly suppressed for native σ⁷⁰ expression) carrying the P_exsC-lacZ (B) or P_exsD-lacZ (C) transcriptional reporter and the p2UY21 ExsA expression plasmid was transformed with a wild-type σ⁷⁰ expression plasmid or an σ⁷⁰ expression plasmid carrying the indicated mutations in region 4.2. The resulting strains were grown in LB to an OD₆₀₀ of 0.6 and assayed for β-galactosidase activity. The reported values (percentage of activity in the presence of wild-type σ⁷⁰ subunit) are the averages from three independent experiments, and error bars represent the standard errors of the means.

FIG. 3.

ExsA-dependent in vitro transcription is dependent on region 4.2 of σ⁷⁰ from P. aeruginosa. (A) Silver-stained SDS-polyacrylamide gel of purified and reconstituted core polymerase subunits α, β, and β′ (lane 1), native σ⁷⁰ (lane 2); σ⁷⁰ carrying the K597A, R596A, and R599A amino acid substitutions (lane 3); and σ⁷⁰ lacking region 4.2 (lane 4). (B and C) Single-round in vitro transcription assays. ExsA_His (35 nM) was incubated with 2 nM supercoiled P_exsC or P_exsD promoter template (pOM90-P_exsC or pOM90-P_exsD) at 25°C in the presence of rATP and rGTP. After 10 min, P. aeruginosa core RNAP, σ⁷⁰-RNAP, or σ⁷⁰ (K597A/R596A/R599A)-RNAP was added (25 nM each; the activity of σ-saturated enzymes was normalized with P_trc), and the reaction mixture was incubated for 1 min at 25°C. Heparin and substrate nucleotides (including 2.5 μCi [α-³²P]CTP) were immediately added, and the reaction mixture was incubated for 5 min at 25°C. Reactions were terminated, and the resulting products were electrophoresed on a 5% denaturing polyacrylamide-urea gel and subjected to phosphorimaging. The ExsA-dependent terminated transcripts (261 nt) from the P_exsC or P_exsD promoter and the runoff transcripts (250 or 180 nt) from the P_trc promoter are indicated.

FIG. 4.

The near-consensus −35 hexamer in the P_exsC promoter is not required for ExsA-independent transcription. (A) Diagram showing the mutant P_exsC promoter derivatives used in this experiment. The −35, extended −10, and −10 elements are boxed, and the individual point mutations are in bold. (B) Single-round in vitro transcription assays showing ExsA-independent transcription from P_exsC derivatives containing −35 (G41T, T40A, G39C, A38T, C37G, A36T, and A33G), extended −10 (TG), and −10 (T8G) point mutations. Reactions were performed as described in the legend to Fig. 3, except open complexes were allowed to form for 20 min in the absence of ExsA. (C) Quantification of the in vitro transcription data shown in panel B. The amount of exsC transcript produced in each experiment was normalized to an ExsA-independent transcript (64) produced from a weak promoter on the minicircle backbone. The reported values are the averages from three independent experiments, and error bars represent the standard errors of the means.

FIG. 5.

The extended −10 element within the P_exsC promoter is important for overall promoter activity independent of ExsA function. (A and B) Single-round in vitro transcription assays and quantification of the corresponding transcripts from the P_exsC, P_exsC-TG, and P_exsCT8G promoters. Experiments were performed as described in the legend to Fig. 3, allowing 1 min for open complex formation in both the absence and presence of ExsA. The reported values (arbitrary densitometry units) are the averages from three independent experiments, and error bars represent the standard errors of the mean. (C) Electrophoretic mobility shift assays (EMSAs) of the P_exsC and P_exsC-TG promoter probes. Specific (SP) and nonspecific (Non-SP) probes (0.25 nM each) were incubated in the absence of ExsA_His (−) or with increasing concentrations of ExsA_His (1 to 36 nM; 2-fold dilutions) for 15 min, followed by electrophoresis and phosphorimaging. ExsA_His-dependent shift products 1 and 2 are indicated.

FIG. 6.

Region 4.2 of σ⁷⁰ is not required for ExsA-independent transcription of the P_exsC promoter. (A) Diagram of transcription templates used in this experiment. The −35 regions (underlined), extended −10 elements (boxed), −10 elements (boxed), and point mutations (bold) are indicated. (B) Single-round in vitro transcription assays were performed with σ⁷⁰ and σ^70Δ4.2 reconstituted RNAP holoenzymes normalized for specific activity using the P_RE# extended −10 promoter (lanes 3 and 4). Reactions were performed as described in the legend to Fig. 3, and open complexes were allowed to form for 1 min (lanes 1 to 4, 9, and 10) or 20 min (lanes 5 to 8) as indicated.