A two gene, two-stage system mediates the defense of E. Coli in response to superoxide. The second stage is carried out by SoxS, a member of the AraC/XyIS family of DNA binding proteins and the direct transcriptional activator of the soxRS regulon. SoxS possesses a number of features that make it a potentially important regulatory protein for further study: SoxS is small, only 107 amino acid residues in length; SoxS binds DNA as a monomer and has no know ligand; the consensus 'soxbox' binding site of SoxS is highly degenerate and lacks any obvious symmetry; SoxS is an 'ambidextrous' transcriptional activator, activation requiring the C-terminal domain (CTD) of the RNA polymerase alpha subunit for activation at the zwf and fpr promoters but neither the alpha CTD nor the CTD of the sigma subunit at the fumC, micF, nfo, or sodA promoters; SoxS activation from the zwf and fpr soxboxes displays a strict positional dependence. Moreover, because SoxS is closely related (50% amino acid identity) to the MarA, Rob, and TetD proteins, information gained from the study of SoxS will contribute to the understanding of the other three proteins. The long term objective of this proposal is to elucidate the structure- function relationships of SoxS as a model monomeric transcriptional activator that functions without a ligand.
Specific Aim 1 is to determine the consensus DNA recognition sequence of SoxS. This will be accomplished by selecting tight binding sequences from a pool of random oligonucleotides, by using the challenge phage system to isolate mutant sites unable to bind SoxS, and by hydroxyl radical interference assays of SoxS mutants that are able to bind soxboxes with various mutations; the mutants will be characterized by DNA sequencing and in vitro transcription experiments employing mutants SoxS proteins purified as fusions to Maltose Binding Protein.
Specific Aim 3 is to identify the amino acid residues of SoxS involved in transcriptional activation at the two classes of promoters. This will be accomplished by the isolation of positive control mutants of zwf, which will be tested for activation of fumC, and vice versa; the mutants will be characterized by DNA sequencing and in vitro transcription experiments.
Specific Aim 4 is to determine the domain (s)/subunits(s) of RNA polymerase that contact SoxS in activation of the fumC, micF, nfo of sodA promoters. This will be accomplished by systematic mutageneses of each subunit and a genetic screen for specific defects in activation.
|Taliaferro, Lanyn P; Keen 3rd, Edward F; Sanchez-Alberola, Neus et al. (2012) Transcription activation by Escherichia coli Rob at class II promoters: protein-protein interactions between Rob's N-terminal domain and the ?(70) subunit of RNA polymerase. J Mol Biol 419:139-57|
|Zafar, M Ammar; Sanchez-Alberola, Neus; Wolf Jr, Richard E (2011) Genetic evidence for a novel interaction between transcriptional activator SoxS and region 4 of the ýý(70) subunit of RNA polymerase at class II SoxS-dependent promoters in Escherichia coli. J Mol Biol 407:333-53|
|Zafar, M Ammar; Shah, Ishita M; Wolf Jr, Richard E (2010) 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. J Mol Biol 401:13-32|
|Griffith, Kevin L; Fitzpatrick, M Megan; Keen 3rd, Edward F et al. (2009) Two functions of the C-terminal domain of Escherichia coli Rob: mediating ""sequestration-dispersal"" as a novel off-on switch for regulating Rob's activity as a transcription activator and preventing degradation of Rob by Lon protease. J Mol Biol 388:415-30|
|Shah, Ishita M; Wolf Jr, Richard E (2006) Sequence requirements for Lon-dependent degradation of the Escherichia coli transcription activator SoxS: identification of the SoxS residues critical to proteolysis and specific inhibition of in vitro degradation by a peptide comprised of the N-terminal J Mol Biol 357:718-31|
|Shah, Ishita M; Wolf Jr, Richard E (2006) Inhibition of Lon-dependent degradation of the Escherichia coli transcription activator SoxS by interaction with 'soxbox' DNA or RNA polymerase. Mol Microbiol 60:199-208|
|Griffith, Kevin L; Becker, Stephen M; Wolf Jr, Richard E (2005) Characterization of TetD as a transcriptional activator of a subset of genes of the Escherichia coli SoxS/MarA/Rob regulon. Mol Microbiol 56:1103-17|
|Shah, Ishita M; Wolf Jr, Richard E (2004) Novel protein--protein interaction between Escherichia coli SoxS and the DNA binding determinant of the RNA polymerase alpha subunit: SoxS functions as a co-sigma factor and redeploys RNA polymerase from UP-element-containing promoters to SoxS-dependent p J Mol Biol 343:513-32|
|Griffith, Kevin L; Wolf Jr, Richard E (2004) Genetic evidence for pre-recruitment as the mechanism of transcription activation by SoxS of Escherichia coli: the dominance of DNA binding mutations of SoxS. J Mol Biol 344:1-10|
|Griffith, Kevin L; Shah, Ishita M; Wolf Jr, Richard E (2004) Proteolytic degradation of Escherichia coli transcription activators SoxS and MarA as the mechanism for reversing the induction of the superoxide (SoxRS) and multiple antibiotic resistance (Mar) regulons. Mol Microbiol 51:1801-16|
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