Escherichia coli Rob, SoxS and MarA are monomeric members of the AraC/XylS family of transcription activators, possessing two helix-turn-helix motifs that are used for DNA binding and transcription activation. SoxS and MarA are synthesized de novo in response to superoxide stress and salicylate. Rob is unique, being constitutively expressed at 5,000-10,000 molecules per cell, but maintained in an inactive form by being sequestered into 3-4 intracellular aggregates. Dipyridyl (DIP) and bile salts (DEC) induce Rob activity, by dispersing the aggregates into monomers that activate transcription of 40-50 genes that mediate the cell's defense response against the above stresses. Rob's activity is controlled by sequestration-dispersal (S-D), a new mechanism of induction where under normal conditions Rob resides in an inactive, sequestered state, primed to be dispersed in response to environmental stress. Our goal is to determine how Rob is sequestered and whether a cytoplasmic factor converts dispersed, active Rob into its inactive, sequestered form. In this study, Rob was tagged with Protein A or His₁₀ and protein complex immunoprecipitation was carried out to isolate complexes between Rob and the putative factor (Chapter IV). Potential protein partners of Rob were then identified by mass spectrometry. In addition, we conducted alanine-scanning mutagenesis of Rob's C-terminal domain (CTD), the portion of the protein shown previously to be necessary and sufficient for S-D (Chapter II). In previous work, several regions important in aggregation and induction were identified using quad alanine-scanning mutagenesis. In this work, single alanine mutants of these regions were made in order to identify the amino acids important to the formation or maintenance of the aggregates or to inducer binding. Several mutant phenotypes of Rob were observed: constitutive, uninducible, partially constitutive/inducible and super-aggregated/inducible. Furthermore, direct protein-protein interactions between Rob and RNAP were identified at class II promoters (Chapter V). Moreover, we determined Rob's orientation at class II promoters and demonstrated that Rob occludes σ⁷⁰ region 4 from binding the -35 hexamer at class II promoters (Chapter V). Finally, Rob was shown to form in vivo binary complexes with RNAP, which supports pre-recruitment as Rob's mechanism of transcription activation (Chapter III)
