The Escherichia coli transcriptional activator SoxS is a member of the AraC/XylS family of bacterial regulatory proteins. A two-stage, two-component process is responsible for inducing the member genes of the soxRS regulon. In response to a stress signal emanating from reactive oxygen species or a reduction in the ratio of NADPH or NADH to the corresponding oxidized coenzyme that is generated by redox-cycling compounds, constitutively expressed SoxR induces the de novo synthesis of SoxS. Newly synthesized SoxS in turn activates transcription of ~16 genes that provide protection against oxidative stress.

SoxS is the smallest member of the AraC/XylS family, only 107 amino acids in length, binds as a monomer, bends DNA 35∞, and recognizes a highly degenerate, 20 bp asymmetric DNA site termed a ‘soxbox’. Two classes of SoxS-dependent promoters exist, Class I promoters where the binding site is located upstream of the –35 hexamer in two possible orientations and Class II promoters where the binding site actually overlaps the –35 hexamer. Highly related transcriptional activators MarA and Rob activate transcription from the same set of genes but do so to varying extents. These proteins belong to a subgroup of the AraC/XylS family, sharing 43-59% amino acid sequence identity over the length of SoxS. Expression of these regulatory proteins not only provides protection against oxidative stress but also confers tolerance to organic solvents and resistance heavy metals and various antibiotics.

A highly degenerate binding site suggests the genome contains a large number of SoxS binding sites. Searching the E. coli genome revealed ~12,500 potential SoxS binding sites. Fast-growing cells with 4-6 genomes would then have ~65,000 soxboxes. Western blot analysis determined 2,500 molecules of SoxS accumulate in response to the redox-cycling compound paraquat. This raises the question of how SoxS can locate functional soxboxes when the number of nonfunctional sites far exceeds it, especially given the relatively low number of SoxS molecules. The evidence suggests a new mechanism of transcription activation, pre-recruitment. Pre-recruitment suggests SoxS first interacts with RNA polymerase in solution and then scans the chromosome for functional SoxS-dependent binding sites.

The main focus of my research is to determine how SoxS and Rob interact with the transcriptional machinery to activate their target genes. I will identify the residues involved in protein-protein interactions between SoxS and RNA polymerase using UV-induced photocrosslinking. In addition to photocrosslinking, I will use a molecular genetics approach to determine which residues of the RNA polymerase a-CTD and s70 region 4 are important for Rob-dependent activation.

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