Protein-Protein Interactions Between σ70 Region 4 of RNA Polymerase and Escherichia coli SoxS a Transcription Activator that Functions by the Pre-recruitment Mechanism: Evidence for Off-DNA and On-DNA Interactions

摘要

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 protein-protein 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 σ⁷⁰ subunit (σ⁷⁰ R4) of RNA polymerase that reduce SoxS activation of class II promoters. With genetic epistasis tests between these σ⁷⁰ 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 σ⁷⁰ 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 σ⁷⁰ R4 that reduce activation in E. coli also reduce the SoxS-σ⁷⁰ 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 σ⁷⁰ 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.