DNA Binding and Transcriptional Activation by SoxS

描述（由申请人提供）：Escherichia coil的SoxS是转录调节因子AraC/Xy1S家族的成员，其中许多可激活细菌病原体中的毒力因子。在内源性产生超氧化物的氧化还原循环化合物的反应中，组成型表达的SoxR诱导SoxS的合成，进而激活执行防御反应的基因的转录。SoxS很小，长度只有107个氨基酸，没有配体，作为单体结合到一个高度简并的不对称DNA位点“soxbox”上，并激活两类启动子的转录，一种是结合位点与-35启动子六聚体重叠，另一种是结合位点位于上游，根据位置的不同，有两种可能的方向。亲缘关系密切的家族成员MarA和Rob与SoxS激活相同的一组基因，尽管程度不同。这些基因的表达不仅提供抗氧化防御，而且赋予对各种抗生素的抗性和对有机溶剂的耐受性。

DESCRIPTION (provided by applicant): 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.