DNA Binding and Transcriptional Activation by SoxS

SoxS 的 DNA 结合和转录激活

• 批准号: 6755079
• 负责人: Richard E Wolf
• 金额: $ 33.58万
• 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/6755079
• 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）：大肠杆菌的SoxS是转录调节因子AraC/Xy 1 S家族的成员，其中许多转录调节因子可激活细菌病原体中的毒力因子。响应于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，SoxS继而激活进行防御反应的基因的转录。SoxS很小，长度仅为107个氨基酸，没有配体，作为单体与称为“soxbox”的高度简并的不对称DNA位点结合，并激活两类启动子的转录，一类是结合位点与-35启动子六聚体重叠，另一类是结合位点位于两种可能取向中的任一种的上游，这取决于位置。近亲家庭成员MarA和Rob激活了与SoxS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，还赋予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，转录激活主要通过称为“募集”的过程进行，但也通过“募集后”途径进行。在这里，证据表明，SoxS激活转录的一种新的机制，“预招聘”。在预募集中，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。如果通过进一步的研究证实，这种激活途径将为细胞中SoxS结合位点的数量（~ 50，000）远远超过每个细胞中SoxS分子的数量（~350）这一难题提供解决方案。证据还提出，SoxS本质上是不稳定的，不稳定性提供了一个解释如何SoxRS监管系统重置，一旦压力信号已经消散。最后，有证据表明，抗氧化还原循环化合物的RNA聚合酶的omega亚基的细胞丰度呈负相关。本研究的主要目的是：（1）利用遗传学和生物化学的方法，在体内和体外研究SoxS和RNAP在溶液中的相互作用;（2）研究Ⅰ类和Ⅱ类启动子下SoxS和RNAP之间的相互作用;（3）鉴定降解SoxS的蛋白酶系统，并确定SoxS的不稳定性;和（4）确定RNAP的ω亚基在SoxS的调节和通过SoxS调节中的潜在作用。实现这些目标将为实现全面理解SoxS和相关蛋白MarA和Rob的DNA结合和转录激活特性的长期目标提供重大进展。