DNA Binding and Transcriptional Activation by SoxS

SoxS 的 DNA 结合和转录激活

• 批准号: 6541680
• 负责人: Richard E Wolf
• 金额: $ 34.19万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE COUNTY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-04-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6541680
• 关键词: DNA binding protein; DNA directed RNA polymerase; Escherichia coli; bacterial genetics; binding sites; genetic promoter element; genetic transcription; oxidative stress; protein degradation; protein protein interaction; transcription factor

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.

描述（由申请人提供）：埃希氏菌的 SoxS 是 AraC/Xy1S 转录调节因子家族的成员，其中许多转录调节因子激活细菌病原体中的毒力因子。为了响应内源性产生超氧化物的氧化还原循环化合物，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，作为单体与称为“soxbox”的高度简并、不对称 DNA 位点结合，并激活两类启动子的转录，一类启动子的结合位点与 -35 启动子六聚体重叠，另一类的结合位点位于两种可能方向的上游，具体取决于位置。密切相关的家族成员 MarA 和 Rob 激活与 SoxS 相同的基因组，尽管程度不同。这些基因的表达不仅提供抗氧化防御，还赋予对多种抗生素的抗性和对有机溶剂的耐受性。 多年的基因调控研究表明，转录激活主要通过称为“招募”的过程进行，但也通过“招募后”途径进行。这里提供的证据表明 SoxS 通过一种新机制“预招募”激活转录。在预招募中，新合成的 SoxS 首先与溶液中的 RNA 聚合酶结合，然后 SoxS-RNA 聚合酶二元复合物扫描染色体以寻找 SoxS 依赖性启动子。如果通过进一步研究证实，这种激活途径将为解决细胞中 SoxS 结合位点数量（约 50,000 个）远远超过每个细胞中 SoxS 分子数量（约 350 个）的难题提供解决方案。还有证据表明 SoxS 本质上不稳定；不稳定性解释了压力信号消失后 SoxRS 监管系统如何重置。最后，有证据表明对氧化还原循环化合物的抗性与 RNA 聚合酶 omega 亚基的细胞丰度呈负相关。该提案有四个具体目标：（1）利用遗传和生化方法在体内和体外表征溶液中SoxS和RNAP之间的蛋白质-蛋白质相互作用； (2) 表征 I 类和 II 类启动子处 SoxS 和 RNAP 之间的蛋白质-蛋白质相互作用； (3) 鉴定降解SoxS的蛋白酶系统并确定导致其不稳定的SoxS特性； (4) 确定 RNAP 的 omega 亚基在 SoxS 调节中的潜在作用。实现这些目标将为实现充分了解 SoxS 及相关蛋白 MarA 和 Rob 的 DNA 结合和转录激活特性的长期目标取得重大进展。