DNA BINDING AND TRANSCRIPTIONAL ACTIVATION BY SOXS

SOXS 的 DNA 结合和转录激活

• 批准号: 2838457
• 负责人: Richard E Wolf
• 金额: $ 24.02万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 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/2838457
• 关键词: DNA binding protein; DNA directed RNA polymerase; affinity chromatography; bacteriophage P22; binding sites; chimeric proteins; enzyme structure; gene expression; gene mutation; genetic promoter element; genetic transcription; hydroxyl radical; intermolecular interaction; 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.

E.杆菌 对超氧化物的反应 第二阶段由SoxS进行， DNA结合蛋白的AraC/XyIS家族成员， soxRS调节子的直接转录激活因子。 SoxS拥有 许多功能使其成为潜在的重要监管工具， 进一步研究的蛋白质：SoxS很小，只有107个氨基酸残基 SoxS作为单体结合DNA，没有已知的配体; SoxS的共有“soxbox”结合位点是高度简并的， 任何明显的对称性; SoxS是一个'ambiancus'转录 激活剂，需要C-末端结构域（CTD）的激活 RNA聚合酶α亚基在zwf和fpr处活化 启动子，但既不是α CTD也不是σ亚基的CTD 在fumC、micF、nfo或sodA启动子处; ZWF和FPR SOXbox显示严格的位置依赖性。 此外，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的研究将有助于对其他三个方面的理解 proteins. 这项建议的长远目标，是要阐明有关的架构- SoxS作为单体转录模型的功能关系 在没有配体的情况下起作用的活化剂。具体目标1是确定 SoxS的共有DNA识别序列。 这将是 通过从一个库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分离 突变位点不能结合SoxS，并通过羟基自由基干扰 SoxS突变体的测定，所述突变体能够结合具有各种 突变;突变体将通过DNA测序表征， 使用突变体SoxS蛋白的体外转录实验 纯化为与麦芽糖结合蛋白的融合物。 具体目标3是 鉴定SoxS参与转录的氨基酸残基， 在两类启动子上激活。 这将是完成 通过分离ZWF的阳性对照突变体，将对其进行检测 激活fumC，反之亦然;突变体将 通过DNA测序和体外转录来表征 实验 具体目标4是确定结构域/亚基 与SoxS接触，激活fumC，micF， sodA启动子的nfo。 这将通过系统的 每个亚基的致突变作用和特定缺陷的遗传筛查 激活。