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DNA-Binding Protein Discovery by the Combinatorial Method REPSA

通过组合方法 REPSA 发现 DNA 结合蛋白

基本信息

批准号：
8433672
负责人：
Michael W Van Dyke
金额：
$ 26.98万
依托单位：
KENNESAW STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8433672
关键词：
Affinity Animal Model Bacteria Binding Binding Sites Biochemical Bioinformatics Biological Biological Assay Biological Process Biology Biomedical Research Cells Collaborations DNA DNA Binding DNA Restriction Enzymes DNA Sequence DNA-Binding Proteins Databases Development Escherichia coli Escherichia coli Proteins Fluorescence Polarization Generations Genes Genetic Genome Goals Human Genome Human Genome Project In Vitro Individual Institution Knowledge Laboratories Ligands Maps Methods Molecular Mutate Oligonucleotides Open Reading Frames Organism Orphan Outcome Peptide Mapping Play Protein Binding Proteins Public Health Recombinant Proteins Research Role SECTM1 gene Specificity Technology United States National Institutes of Health Universities combinatorial expectation genetic regulatory protein magnetic beads microbial disease novel public health relevance technology development transcription factor

项目摘要

DESCRIPTION (provided by applicant): Our goal is to advance our understanding of predicted and/or unexpected DNA-binding proteins through the identification of their preferred binding sites by the novel combinatorial approach, Restriction Endonuclease Protection, Selection and Amplification (REPSA). While the Human Genome Project has yielded a wealth of information for both the human genome and other model organisms, much remains to be determined regarding individual genes and the biological roles played by their encoded products. For example, while the bacteria Escherichia coli strain K12 has 4364 open reading frames, only about half of these genes have been well characterized by genetic, biochemical or molecular biological means. Many of the known genes (260+) encode for proteins that presumably bind specific DNA sequences. However, for most of these proteins (>200) their preferred DNA-binding sites have not been determined empirically. We have developed a combinatorial approach, REPSA, which does not require any prior knowledge of a ligand in order to determine its preferred binding site on duplex DNA. Thus we hypothesize that REPSA can be used to identify the preferred DNA- binding sites of uncharacterized proteins in the model organism E. coli K-12. We propose the following four Specific Aims: (1) Identify preferred DNA-binding sites using REPSA with inputs ranging from purified E. coli proteins to bacterial extracts, massively parallel DNA sequencing, and bioinformatics analyses (e.g., Multiple Expectation Maximum for Motif Elicitation, MEME). (2) When necessary, identify proteins bound to identify preferred DNA-binding sites by DNA- affinity/magnetic bead capture and peptide fingerprinting. (3) Validate protein-DNA binding and determine binding specificity using recombinant proteins, point-mutated oligonucleotides, and fluorescence polarization binding assays. (4) Propose potential biological functions for these proteins and their binding sites through binding site mapping on the E. coli genome using Find Individual Motif Occurrence (FIMO) and annotations in available databases (e.g., EcoCyc, EcoliWiki, EcoGene, RegulonDB, UniProtKB). We expect our research to ultimately catalyze numerous studies by us and other laboratories leading to a better understanding of E. coli biology at a molecular level and provide a framework for similar studies in other organisms.

描述（由申请人提供）：我们的目标是通过新的组合方法，限制性内切酶保护，选择和扩增（REPSA），通过鉴定其优选的结合位点，促进我们对预测和/或非预期DNA结合蛋白的理解。虽然人类基因组计划已经为人类基因组和其他模式生物提供了大量信息，但关于单个基因及其编码产物所发挥的生物作用，仍有许多问题有待确定。例如，虽然细菌大肠杆菌菌株K12具有4364个开放阅读框，但这些基因中只有大约一半已经通过遗传、生物化学或分子生物学手段得到了很好的表征。许多已知的基因（260+）编码的蛋白质可能结合特定的DNA序列。然而，对于这些蛋白质中的大多数（>200），它们的优选DNA结合位点尚未根据经验确定。我们已经开发了一种组合的方法，REPSA，它不需要任何配体的先验知识，以确定其优选的双链体DNA上的结合位点。因此，我们假设REPSA可用于鉴定模式生物E. coli K-12。我们提出了以下四个具体目标：（1）使用REPSA确定优选的DNA结合位点，输入范围从纯化的E。大肠杆菌蛋白到细菌提取物，大规模并行DNA测序，和生物信息学分析（例如，模体诱导的多重期望最大值（Multiple Expectation Maximum for Motif Elicitation，MEME）。(2)必要时，通过DNA亲和性/磁珠捕获和肽指纹图谱鉴定结合蛋白，以确定首选DNA结合位点。(3)使用重组蛋白、点突变的寡核苷酸和荧光偏振结合测定法测定结合特异性。(4)通过在E.大肠杆菌基因组中使用查找单个基序出现（FIMO）和可用数据库中的注释（例如，EcoCyc，EcoliWiki，EcoGene，RegulonDB，UniProtKB）。我们希望我们的研究最终能促进我们和其他实验室的大量研究，从而更好地了解E。在分子水平上研究大肠杆菌生物学，并为其他生物体的类似研究提供框架。