DNA Binding and Transcriptional Activation by SoxS

描述(申请人提供)：大肠杆菌的SOXS是转录调节因子AraC/Xy1S家族的成员之一，其中许多转录调节因子激活细菌病原体中的毒力因子。对氧化还原循环化合物内源性产生超氧化物的反应，结构性表达的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，只有107个氨基酸，没有配体，作为单体与高度简并的不对称DNA位点结合，称为Soxbox，并激活来自两类启动子的转录，一类结合位点与-35启动子六角体重叠，另一类结合位点位于上游两个可能的方向之一，取决于位置。密切相关的家族成员玛拉和罗布激活了与袜子相同的一组基因，尽管程度不同。这些基因的表达不仅提供了一种抗氧化防御，而且还赋予了对多种抗生素的抗性和对有机溶剂的耐受性。 多年来对基因调控的研究表明，转录激活主要通过一种称为“招募”的过程进行，但也通过一种“招募后”途径进行。在这里，有证据表明SOXS通过一种新的机制激活转录，即“预招募”。在招募前，新合成的SOXS首先在溶液中与RNA聚合酶结合，然后SOXS-RNA聚合酶二元复合体扫描染色体以寻找SOXS依赖的启动子。如果进一步的研究证实，这一激活途径将为解决细胞内SOXS结合位点的数量(~50,000)远远超过每个细胞的SOXS分子数量(~350)的难题提供解决方案。还提出了SOXS本质上不稳定的证据；不稳定性为SOXRS调控系统如何在压力信号消散后重新启动提供了解释。最后，有证据表明，对氧化还原循环化合物的抵抗力与RNA聚合酶omega亚单位的细胞丰度成反比。这项建议有四个具体目标：(1)利用遗传学和生物化学方法表征SOXS和RNAP在溶液中的蛋白质-蛋白质相互作用；(2)表征SOXS和RNAP在I类和II类启动子上的蛋白质-蛋白质相互作用；(3)鉴定降解SOXS的蛋白酶系统，并确定导致SOXS不稳定的SOXS的性质；以及(4)确定RNAP的omega亚单位在SOXS调控和调控中的潜在作用。实现这些目标将为实现全面了解SOXS及其相关蛋白MarA和Rob的DNA结合和转录激活特性这一长期目标提供重大进展。

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.