Protein-protein interactions between sigma(70) region 4 of RNA polymerase and Escherichia coli SoxS, a transcription activator that functions by the prerecruitment mechanism: evidence for "off-DNA" and "on-DNA" interactions

Abstract

According to the prerecruitment hypothesis, Escherichia coli SoxS activates the transcription of the genes of the SoxRS regulon by forming binary complexes with RNA polymerase (RNAP) that scan the chromosome for class I and class II SoxS-dependent promoters. We showed previously that the alpha subunit's C-terminal domain plays a role in activating both classes of promoter by making protein-protein contacts with SoxS; some of these contacts are made in solution in the absence of promoter DNA, a critical prediction of the prerecruitment hypothesis. Here, we identified seven single-alanine substitutions of the region 4 of sigma(70) (sigma(70) R4) of RNAP that reduce SoxS activation of class II promoters. With genetic epistasis tests between these sigma(70) R4 mutants and positive control mutants of SoxS, we identified 10 pairs of amino acids that interact with each other in E. coli. Using the yeast two-hybrid system and affinity immobilization assays, we showed that SoxS and sigma(70) R4 can interact in solution (i.e., "off-DNA"). The interaction requires amino acids of the class I/II (but not the class II) positive control surface of SoxS, and five amino acids of sigma(70) R4 that reduce activation in E. coli also reduce the SoxS-sigma(70) R4 interaction in yeast. One of the epistatic interactions that occur in E. coli also occurs in the yeast two-hybrid system (i.e., off-DNA). Importantly, we infer that the five epistatic interactions occurring in E. coli that require an amino acid of the class II surface occur "on-DNA" at class II promoters. Finding that SoxS contacts sigma(70) R4 both off-DNA and on-DNA is consistent with the prerecruitment hypothesis. Moreover, SoxS is now the first example of an E. coli transcriptional activator that uses a single positive control surface to make specific protein-protein contacts with two different subunits of RNAP.

Figure 1

Representative epistatic and non-epistatic interactions between alanine substitutions of σ⁷⁰ R4 and alanine substitutions of SoxS that individually confer a defect in transcription activation at a given SoxS-dependent promoter. The experiments were carried out as described in Materials and Methods. A. The left panel shows that at the fumC promoter amino acid substitution R599A of σ⁷⁰ R4 is epistatic to amino acid substitution D9A of SoxS, while the right panel shows that R599A is not epistatic to D79A of SoxS. B. The left panel shows that at the micF promoter, amino acid substitution R603A of σ⁷⁰ R4 is epistatic to amino acid substitution D75A of SoxS, while the right panel shows that R603A is not epistatic to M78A of SoxS. C. The left panel shows that at the zwf promoter amino acid substitution D612A of σ⁷⁰ R4 is epistatic to amino acid substitution K30A, while the right panel shows that D612A is not epistatic to S31A of SoxS.

Figure 2

AIA of the protein–protein interactions between SoxS and amino acids 541–613 of σ⁷⁰ R4. The immobilization of his₆-σ⁷⁰ R4 on Ni-NTA resin and its incubation with a cell-free extract containing wild type SoxS were carried out as described in Materials and Methods, as was the subsequent SDS-PAGE and western blotting of the respective fractions obtained after application of the SoxS-containing extract. FT, flow-through fraction; W3, third wash fraction; EL, eluate. Arrows indicate the location of his₆-σ⁷⁰ R4 and SoxS. A. The blot probed with antibody directed against the his₆ tag. Samples FT and W3 show that all of the σ⁷⁰ R4 bound to the column. Sample EL shows that all of the σ⁷⁰ R4 was eluted from the Ni-NTA resin upon incubation with 0.5 M imidazole. B. The blot probed with anti-SoxS antibodies. The FT sample shows that a large portion of SoxS was not bound to the column while the W3 fraction reveals that all of the non-specifically bound SoxS was washed away. Sample EL shows that SoxS was eluted from the Ni-NTA resin column with 0.5 M imidazole. Taken together, the two blots suggest that σ⁷⁰ R4 binds the column, that the bound protein retains SoxS, and that both proteins are co-eluted.

Figure 3

Effects in the Y2H system of single alanine substitutions of σ⁷⁰ R4 that cause a defect in transcription activation at SoxS-dependent promoters in E. coli. Cultures of strain EGY48[p8oplacZ] carrying pGILDA-SoxS and pB42AD-σ⁷⁰ R4 with wild type and mutant alleles of σ⁷⁰ R4 were grown under inducing conditions and assayed for β-galactosidase activity as described in Materials and Methods. The specific activity of the strain expressing wild type σ⁷⁰ R4 was taken as 100% and the activity values of the strains expressing the mutant alleles are given relative to wild type. As depicted at the bottom of the figure, cell extracts of each culture were subjected to western blotting with antibodies against the HA tag fused to B42AD-σ⁷⁰ R4 and antibodies against β-tubulin, which served as an internal loading control.

Figure 4

Effects in the Y2H system of single alanine substitutions of the class I/II surface of SoxS that confer defects in transcription activation at SoxS-dependent promoters and which display epistasis in E. coli with alanine substitutions of σ⁷⁰ R4. Cultures of strain EGY48[p8oplacZ] carrying pB42AD-σ⁷⁰ R4 and pGILDA-SoxS with wild type and mutant alleles of SoxS were grown under inducing conditions and assayed for β-galactosidase activity as described in Materials and Methods. The specific activity of the strain expressing wild type SoxS was taken as 100% and the activity values of the strains expressing the mutant alleles are given relative to the wild type. Cell extracts of each culture were subjected to western blotting with affinity-purified antibodies against SoxS and antibodies against β-tubulin, which served as an internal loading control.

Figure 5

Representative epistatic and non-epistatic interactions in the Y2H system between alanine substitutions of SoxS and alanine substitutions of σ⁷⁰ R4 that individually confer a defect in transcription activation of the lacZ reporter in the Y2H system. Cultures of strain EGY48[p8oplacZ] carrying plasmid pGILDA-SoxS with wild type and mutant alleles of SoxS and plasmid p42AD-σ⁷⁰ R4 with wild type and mutant alleles of σ⁷⁰ R4 were grown under inducing conditions and assayed for β-galactosidase activity as described in Materials and Methods. The specific activity of the strain expressing wild type SoxS and wild type σ⁷⁰ R4 was taken as 100% and the activity values of the strains expressing the various mutant alleles are given relative to the wild type. An epistatic interaction was observed between σ⁷⁰ substitution D612A and SoxS K30A, a mutant of the class I/II positive control surface. A two tailed t-test was carried out to determine whether the values for the double mutants are significantly different from the values for the corresponding single mutants. The value for the SoxS-K30A + σ⁷⁰ D612A double mutant was not significantly different than that of the single mutant SoxS + σ⁷⁰ D612A (p>0.05) and thus this interaction is epistatic; in contrast, the value for the SoxS-S31A + σ⁷⁰ D612A double mutant was significantly different than that of the single mutant SoxS + σ⁷⁰ D612A (p<0.05) and thus this interaction is non-epistatic.

Figure 6

AIA of the protein–protein interactions between SoxS and full-length σ⁷⁰ The immobilization of wild-type σ⁷⁰ on Ni-NTA resin, the application of cell-free extracts containing wild type and SoxS proteins with multiple positive control substitutions, the recovery of the FT, W3 and EL fractions and western blotting of the fractions with antibodies directed against SoxS and the his₆ tag of σ⁷⁰ were carried out as described in Materials and Methods.