DNA BINDING AND TRANSCRIPTIONAL ACTIVATION BY SOXS

SOXS 的 DNA 结合和转录激活

• 批准号: 2608769
• 负责人: Richard E Wolf
• 金额: $ 23.33万
• 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 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2608769
• 关键词: DNA binding protein; DNA directed RNA polymerase; affinity chromatography; bacteriophage P22; chimeric proteins; enzyme structure; gene expression; gene mutation; genetic promoter element; genetic transcription; hydroxyl radical; intermolecular interaction; molecular site; mutant; nucleic acid sequence; oligonucleotides; polymerase chain reaction; protein purification; protein sequence; protein structure function

A two gene, two-stage system mediates the defense of E. Coli in response to superoxide. The second stage is carried out by SoxS, a member of the AraC/XyIS family of DNA binding proteins and the direct transcriptional activator of the soxRS regulon. SoxS possesses a number of features that make it a potentially important regulatory protein for further study: SoxS is small, only 107 amino acid residues in length; SoxS binds DNA as a monomer and has no know ligand; the consensus 'soxbox' binding site of SoxS is highly degenerate and lacks any obvious symmetry; SoxS is an 'ambidextrous' transcriptional activator, activation requiring the C-terminal domain (CTD) of the RNA polymerase alpha subunit for activation at the zwf and fpr promoters but neither the alpha CTD nor the CTD of the sigma subunit at the fumC, micF, nfo, or sodA promoters; SoxS activation from the zwf and fpr soxboxes displays a strict positional dependence. Moreover, because SoxS is closely related (50% amino acid identity) to the MarA, Rob, and TetD proteins, information gained from the study of SoxS will contribute to the understanding of the other three proteins. The long term objective of this proposal is to elucidate the structure- function relationships of SoxS as a model monomeric transcriptional activator that functions without a ligand. Specific Aim 1 is to determine the consensus DNA recognition sequence of SoxS. This will be accomplished by selecting tight binding sequences from a pool of random oligonucleotides, by using the challenge phage system to isolate mutant sites unable to bind SoxS, and by hydroxyl radical interference assays of SoxS mutants that are able to bind soxboxes with various mutations; the mutants will be characterized by DNA sequencing and in vitro transcription experiments employing mutants SoxS proteins purified as fusions to Maltose Binding Protein. Specific Aim 3 is to identify the amino acid residues of SoxS involved in transcriptional activation at the two classes of promoters. This will be accomplished by the isolation of positive control mutants of zwf, which will be tested for activation of fumC, and vice versa; the mutants will be characterized by DNA sequencing and in vitro transcription experiments. Specific Aim 4 is to determine the domain (s)/subunits(s) of RNA polymerase that contact SoxS in activation of the fumC, micF, nfo of sodA promoters. This will be accomplished by systematic mutageneses of each subunit and a genetic screen for specific defects in activation.

两个基因、两个阶段的系统介导大肠杆菌的防御 对超氧化物的反应。 第二阶段由 SoxS 执行， DNA 结合蛋白 AraC/XyIS 家族的成员 soxRS 调节子的直接转录激活剂。 SoxS 拥有 许多特征使其成为潜在的重要监管 待进一步研究的蛋白质：SoxS很小，只有107个氨基酸残基 长度； SoxS 作为单体结合 DNA，并且没有已知的配体；这 SoxS 的共有“soxbox”结合位点高度简并并且缺乏 任何明显的对称性； SoxS 是一种“灵巧”转录 激活剂，激活需要 C 端结构域 (CTD) 用于激活 zwf 和 fpr 的 RNA 聚合酶 α 亚基 启动子，但既不是 alpha CTD 也不是 sigma 亚基的 CTD 在 fumC、micF、nfo 或 sodA 启动子处； SoxS 激活来自 zwf 和 fpr soxboxes 显示出严格的位置依赖性。 而且，由于SoxS密切相关（50%氨基酸同一性） 对于 MarA、Rob 和 TetD 蛋白，从 对 SoxS 的研究将有助于理解其他三个 蛋白质。 该提案的长期目标是阐明结构 - SoxS 作为单体转录模型的功能关系 无需配体即可发挥作用的激活剂。具体目标 1 是确定 SoxS 的共有 DNA 识别序列。 这将是 通过从池中选择紧密结合序列来完成 随机寡核苷酸，通过使用挑战噬菌体系统来分离 突变位点无法结合SoxS，并受到羟基自由基干扰 能够结合 soxbox 的 SoxS 突变体的测定 突变；突变体将通过 DNA 测序进行表征 使用突变体 SoxS 蛋白的体外转录实验 纯化为麦芽糖结合蛋白的融合物。 具体目标 3 是 鉴定参与转录的 SoxS 氨基酸残基 两类启动子的激活。 这将实现 通过分离 zwf 的阳性对照突变体，对其进行测试 用于激活 fumC，反之亦然；突变体将是 以 DNA 测序和体外转录为特征 实验。 具体目标 4 是确定结构域/亚基 RNA聚合酶在fumC、micF的激活中接触SoxS， sodA 启动子的信息。 这将通过系统地完成 每个亚基的诱变和针对特定缺陷的遗传筛查 激活中。