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.

一种双基因、两阶段系统介导的E.Coli防御 对超氧化物的反应。第二阶段由SOXS执行，一个 AraC/XyIS DNA结合蛋白家族的成员 SOXRS调节子的直接转录激活剂。索克斯拥有 许多功能使其成为潜在的重要监管机构 进一步研究的蛋白质：SOXS很小，只有107个氨基酸残基 在长度上；SOXS以单体形式结合DNA，并且没有已知的配体； Soxs共识的Soxbox结合位点高度退化，缺乏 任何明显的对称性；Soxs是一种“双灵巧”转录 激活剂，需要C-末端结构域(CTD)的激活 在zwf和fpr激活的RNA聚合酶α亚单位 启动子，但既不是αCTD，也不是Sigma亚单位的CTD 在FumC、MICF、NFO或苏打促进剂；SOXS从 ZWF和FPR SOXBOX表现出严格的位置依赖关系。 此外，因为SOXS是密切相关的(50%的氨基酸同一性) 对于Mara、Rob和TetD蛋白，从 对SOX的研究将有助于理解其他三个 蛋白质。 这项建议的长期目标是澄清以下结构- 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激活剂。具体目标1是确定 SOXS的一致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，用挑战噬菌体系统分离 不能结合SOX的突变位点，并受羟基自由基干扰 Soxs突变体能够与不同种类的Soxbox结合 突变；突变体将通过DNA测序和 突变型SoxS蛋白的体外转录实验 纯化后与麦芽糖结合蛋白融合。具体目标3是 鉴定参与转录的SOXS的氨基酸残基 两类启动子的激活。这将会实现的 通过对zwf阳性对照突变体的分离，对其进行检测。 用于激活fumC，反之亦然；突变体将 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(S)/亚基(S) 在激活fumC，MICF， 苏打促进剂的NFO。这将通过系统化的 每个亚基的突变和特定缺陷的遗传筛查 在激活中。