DNA BINDING AND TRANSCRIPTIONAL ACTIVATION BY SOXS

SOXS 的 DNA 结合和转录激活

• 批准号: 2021825
• 负责人: Richard E Wolf
• 金额: $ 24.18万
• 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/2021825
• 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.

两个基因的两个阶段系统介导了大肠杆菌的防御 对超氧化物的反应。 第二阶段是由Soxs进行的 DNA结合蛋白的ARAC/XYIS家族的成员和 SOXRS调节的直接转录激活剂。 袜子拥有 许多功能使其成为潜在的重要调节 进一步研究的蛋白质：Soxs很小，只有107个氨基酸残基 长度； Soxs将DNA作为单体结合，并且不知道配体。这 Soxs的共识“ Soxbox”结合位点高度退化，缺乏 任何明显的对称性； Soxs是一种“灵巧”的转录 激活剂，激活需要C末端结构域（CTD） RNA聚合酶α亚基在ZWF和FPR上激活 启动子，但既不是Alpha CTD和Sigma亚基的CTD 在FUMC，MICF，NFO或苏打启动子上； Soxs激活 ZWF和FPR SOXBOXES显示出严格的位置依赖性。 此外，由于SOXS密切相关（50％氨基酸身份） 到Mara，Rob和TETD蛋白，从中获得的信息 袜的研究将有助于对其他三个的理解 蛋白质。 该提案的长期目标是阐明结构 - SOX作为模型单体转录的功能关系 活化剂在没有配体的情况下起作用。具体目标1是确定 SOXS的共识DNA识别序列。 这将是 通过从一个池中选择紧密的绑定序列来完成 随机寡核苷酸，通过使用挑战噬菌体系统隔离 突变位点无法结合Soxs和羟基干扰 能够将Soxboxes绑定到各种的Soxs突变体的测定 突变；突变体将以DNA测序和 使用突变体Soxs蛋白的体外转录实验 纯化为融合麦芽糖结合蛋白。 具体目标3是 识别参与转录的Sox的氨基酸残基 在两个类别的启动子上激活。 这将完成 通过隔离ZWF的阳性对照突变体，将进行测试 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。 特定目的4是确定域/亚基（S） RNA聚合酶在FUMC激活中接触Soxs的RNA聚合酶 苏打启动子的NFO。 这将通过系统 每个亚基的诱变和特定缺陷的遗传筛查 在激活中。