Protein–Protein Interactions Between σ70 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 pre-recruitment hypothesis, Escherichia coli SoxS activates transcription of the genes of the SoxRS regulon by forming binary complexes with RNA polymerase that scan the chromosome for class I and class II SoxS-dependent promoters. Previously, we showed that the a subunit's C-terminal domain plays a role in activating both classes of promoter by making proteinprotein contacts with SoxS; some of these contacts are made in solution in the absence of promoter DNA, a critical prediction of the pre-recruitment hypothesis. Here, we identified seven single alanine substitutions of region 4 of the σ 70 subunit (σ 70 R4) of RNA polymerase that reduce SoxS activation of class II promoters. With genetic epistasis tests between these σ 70 R4 mutants and positive control mutants of SoxS we identified ten 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 σ 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 σ 70 R4 that reduce activation in E. coli also reduce the SoxS-σ 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 σ 70 R4 both off-DNA as well as on-DNA is consistent with the pre-recruitment 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 RNA polymerase.

Genetic Evidence for a Novel Interaction between Transcriptional Activator SoxS and Region 4 of the σ70 Subunit of RNA Polymerase at Class II SoxS-Dependent Promoters in Escherichia coli

“…As expected, none of the σ 70 R4 substitutions reduced activation of the canonical class I promoter, fpr , because its soxbox is too far (15 bp) from σ 70 R4 to interact with it56. Furthermore, we determined that SoxS and σ 70 R4 also interact in solution in the absence of specific DNA binding and that amino acids of the class I/II surface of SoxS are required for these “off-DNA” interactions56.…”

“…Previous work has shown that SoxS and σ 70 interact with one another and that amino acids in the distal portion of region 4.2 (residues 590–600) and the C-terminal tail (residues 591–612) are important for SoxS-dependent transcription activation of class II promoters fumC and micF and the non-canonical class I zwf promoter but not the class I fpr promoter56. Here, we investigated whether amino acids of σ 70 that reside in the distal portion of region 3.2 (residues 531–540), in region 4.1 (residues 541–570) and in the N-terminal portion of region 4.2 (residues 571–590)4; 5; 10 play a role in transcription from SoxS-dependent promoters.…”

“…Furthermore, we determined that SoxS and σ 70 R4 also interact in solution in the absence of specific DNA binding and that amino acids of the class I/II surface of SoxS are required for these “off-DNA” interactions56. In conclusion, these experiments provide evidence that the class I/II surface of SoxS makes protein-protein contacts with domains of two different subunits of RNAP, the α-CTD and σ 70 R4, with some interactions likely to occur off-DNA and others on-DNA and/or to be dependent on the specific promoter being activated55; 56.…”

Residue Substitutions Near the Redox Center of Bacillus subtilis Spx Affect RNA Polymerase Interaction, Redox Control, and Spx-DNA Contact at a Conserved cis-Acting Element

“…Introduction of a second substitution at R92 does not change the level of mutant Spx activity in vivo, indicating that C10A is epistatic to R92A, and the mutant phenotype of R92A is observed only when a disulfide can be generated. Such epistasis experiments using multiple residue substitutions has been applied to uncover protein-protein contact surfaces on SoxS and its binding partner, 70 of E. coli RNAP (46,47). It is not clear how the R92 side chain transduces a redox signal from the disulfide center to the target-interacting surfaces of Spx and ultimately to RNAP.…”

Global transcriptome analysis of the E. coli O157 response to Agrimonia pilosa extract

“…SoxS is the direct transcriptional activator of the E. coli superoxide (SoxRS) regulon 31 . But the RecQ family of DNA helicases leading to the induction of the member gene of the regulon during oxidative stress is eccentric in that it occurs in two temporary stages of transcription: the first mediated by soxR and the second by soxS 31,32 . SoxS synthesized de novo in reaction to the oxidative stress, mounts the defense reaction by activating the transcription of the w40 genes of the regulon 33 .…”