SoxS of Escherichia coil is a member of the AraC/Xy1S family of transcription regulators, many of which activate virulence factors in bacterial pathogens. In response to redox-cycling compounds that endogenously generate superoxide, constitutively expressed SoxR induces synthesis of SoxS, which in turn activates transcription of the genes that carry out the defense response. SoxS is small, only 107 amino acids in length, has no ligand, binds as a monomer to a highly degenerate, asymmetric DNA site termed """"""""soxbox"""""""", and activates transcription from two classes of promoter, one where the binding site overlaps the -35 promoter hexamer, and the other where the binding site lies upstream in either of two possible orientations, depending on the position. Closely related family members MarA and Rob activate the same set of genes as SoxS, albeit to different degrees. Expression of these genes not only provides an antioxidant defense but also confers resistance to diverse antibiotics and tolerance to organic solvents. ? ? Years of study of gene regulation have shown that transcription activation proceeds mainly by a process known as """"""""recruitment"""""""" but also by a """"""""post-recruitment"""""""" pathway. Here, evidence is presented indicating that SoxS activates transcription by a new mechanism, """"""""pre-recruitment"""""""". In pre-recruitment, newly synthesized SoxS first binds to RNA polymerase in solution and then the SoxS-RNA polymerase binary complex scans the chromosome in search of SoxS-dependent promoters. This activation pathway, if substantiated by further study, would provide a solution to the conundrum that the number of SoxS binding sites in the cell (~50,000) far exceeds the number of SoxS molecules per cell (~350). Evidence is also presented that SoxS is intrinsically unstable; instability provides an explanation for how the SoxRS regulatory system resets once the stress signal has dissipated. Lastly, evidence is presented that resistance to redox-cycling compounds is inversely related to the cellular abundance of the omega subunit of RNA polymerase. This proposal has four specific aims: (1) characterize the protein-protein interactions between SoxS and RNAP in solution both in vivo and in vitro using genetic and biochemical methods; (2) characterize the protein-protein interactions between SoxS and RNAP at class I and class II promoters; (3) identify the protease system that degrades SoxS and determine the properties of SoxS that contribute to its instability; and (4) determine the potential role of the omega subunit of RNAP in regulation of and by SoxS. Achieving these aims will provide significant progress toward accomplishing the long term objective of understanding fully the DNA binding and transcription activation properties of SoxS and related proteins MarA and Rob.
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