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）：Escherichia Coil的Sox是ARAC/XY1S转录调节剂家族的成员，其中许多是激活细菌病原体中毒力因子的成员。响应于内源产生超氧化物的氧化还原循环化合物，组成型SOXR诱导了SOX的合成，这又激活了执行防御反应的基因的转录。 SOX很小，只有107个氨基酸，没有配体，作为单体结合，与高度退化的，不对称的DNA位点称为“ Soxbox”，并激活了两个启动子的转录，一个启动子中的一个结合位点与-35启动子六角体六角形位点重叠，而其他位置则在两个可能的位置上都在两个可能的位置上，并且在两个可能的位置上都可以依赖于两个可能的位置。密切相关的家庭成员Mara和Rob激活了与Sox相同的基因，尽管在不同程度上。这些基因的表达不仅提供了抗氧化剂防御，而且还赋予对各种抗生素和对有机溶剂的耐受的抗性。 对基因调控的多年研究表明，转录激活主要是通过称为“募集”的过程进行的，也是通过“招募后”途径进行的。在这里，提供了证据，表明Soxs通过一种新的机制“招聘前”激活转录。在恢复前，新合成的SOXS首先与溶液中的RNA聚合酶结合，然后结合SOXS-RNA聚合酶二进制复合物，扫描染色体，以寻找SOXSSSSS依赖性启动子。如果通过进一步研究证实，该激活途径将为难题提供解决方案，即细胞中的Soxs结合位点的数量远超过每个细胞的Soxs分子的数量（〜350）。还有证据表明袜本质上是不稳定的。不稳定性提供了一个解释，一旦应力信号消散，SOXRS调节系统如何重置。最后，有证据表明，对氧化还原循环化合物的抗性与RNA聚合酶的欧米茄亚基的细胞丰度成反比。该提案具有四个具体的目的：（1）使用遗传和生化方法来表征体内和体外溶液中蛋白质 - 蛋白质相互作用； （2）表征了I类和II类启动子Sox和RNAP之间的蛋白质 - 蛋白质相互作用； （3）确定降解Soxs并确定有助于其不稳定性的SOX的特性的蛋白酶系统； （4）确定RNAP欧米茄亚基在Soxs和Soxs的调节中的潜在作用。实现这些目标将为完成完全理解SOXS和相关蛋白质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。