Characterization of On DNA and Off DNA interactions between transcriptional activator SoxS and RNA Polymerase of Escherichia coli.
Creators: name: Zafar, Muhammad Ammar
Publisher: name: Maryland Shared Open Access Repository
Year: 2009
Subjects: genetic epistasis
Oxidative stress
RNAP
SoxRS regulon
Transcription activation
Yeast two hybrid

description: SoxS, a member of AraC/XylS family of transcriptional activators, activates genes of the soxRS regulon involved in providing the cells defense against oxidative stress. SoxS forms a binary complex with RNA polymerase (RNAP) in solution. This complex then binds DNA and activates transcription via a mechanism termed pre-recruitment. This binary complex involves contacts between SoxS and the Alpha C-terminal domain (CTD) and Sigma70 subunits of RNAP. The Alpha CTD of RNAP enhances transcription by contacting transcriptional activators or a promoter element. Work from our lab identified the Alpha CTD as a subunit of RNAP involved in binary complex formation. The 265 DNA binding determinant of the Alpha CTD was identified as being required for making the protein-protein contact between Alpha CTD and SoxS. Previously, the Alpha CTD was found to be required for in vitro transcription activation at class I SoxS-dependent promoters but not required at class II SoxS-dependent promoters. By using an alanine scanning library of the Alpha CTD and additionally by removing it (Alpha-235), I found that Alpha CTD is required at class I and class II SoxS-dependent promoters in vivo. Furthermore, in vitro binding assays confirmed the results observed in vivo, which implicated amino acids of the 265 determinant in transcription activation at SoxS-dependent promoters. Moreover, using genetic epistatsis tests, I identified the interacting partners between the Alpha CTD and SoxS that are involved in these protein-protein interactions. These epistasis experiments revealed an extensive interaction that probably occurs between the Alpha CTD and SoxS on DNA, with both the Alpha CTD subunits being required to activate transcription at class II SoxS-dependent promoters. The Sigma 70 subunit of the RNAP also provides the specificity required for transcription activation, as it recognizes the various promoter elements. Interacting partners between Sigma 70 subunit of the RNAP and AraC/XylS family of transcription activators are known. At a class II SoxS-dependent promoter, SoxS, lies in close proximity to Sigma 70 subunit and possibly involved in important protein-protein interactions leading to transcription activation. By testing Sigma 70 region 4 library of alanine substitutions I was able to determine several Sigma 70 residues that were important in SoxS-dependent activation. By performing genetic epistasis experiments, I was able to identify several putative amino acid residues that were involved in protein-protein contact between SoxS and Sigma 70. Here, I also show that a specific interaction occurs between SoxS and Sigma 70 subunit in vivo in the yeast-two-hybrid assay and in vitro with affinity immobilization assay. This interaction is disrupted by specific SoxS positive control mutants. Class I/II positive control mutants disrupts the interaction in vivo as well as in vitro assays, whereas class II surface mutants only disrupt in E. coli leading us to believe that class II surface is involved in protein-protein contact with Sigma 70 on SoxS promoter DNA whereas class I/II surface mutants play a role in binary complex formation with RNAP subunits in solution as well as at SoxS-dependent promoters. In addition, I wanted to determine the role of Sigma 70 at SoxS-dependent promoters, especially at class II promoters, where SoxS binding site overlaps the -35 hexamer of the promoters. For this purpose, I created an alanine scanning library of the region 4 of Sigma 70. Region 4 is important, as it contacts promoter DNA, the core of RNAP core and it also interacts with other transcriptional regulators. Our library is the first of its kind as it mutagenizes the entire region 4 of Sigma 70. I determined that the interaction between Sigma 70 region 4 and promoter DNA are important at class I SoxS-dependent promoters, whereas at class II SoxS-dependent promoters that Sigma 70 region 4 does not contact the -35 promoter hexmaer. In contrast, at CRP-dependent promoters lac and galP1, our library revealed amino acids important for contacting the respective -35 hexamers.  Finally, using genetic epistasis experiments, I determined the orientation and position of SoxS at SoxS-dependent promoters. These data suggest that SoxS and Sigma 70 interact in a novel fashion at class II SoxS-dependent promoters such that the two proteins do not accommodate one another near the -35 hexamer rather Sigma 70 Region 4 is occluded from the -35 hexamer.