Functional effects of exchanging domains and linkers in transcription regulators

转录调节因子中交换域和连接子的功能效应

• 批准号: 7319211
• 负责人: LISKIN SWINT-KRUSE
• 金额: $ 26.12万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7319211
• 关键词: Address; Affect; Affinity; Agreement; Algorithms; Amino Acid Substitution; Amino Acids; Binding; Binding Sites; Bioinformatics; Biological Assay; Cartoons; Cations; Chimera organism; Chimeric Proteins; Communication; DNA; DNA Binding; DNA Binding Domain; Data; Data Set; Databases; Engineering; Escherichia coli; Family; Family Leave; Family member; Genetic Recombination; Genetic Transcription; Genomics; Goals; Gray unit of radiation dose; Helix (Snails); Homologous Gene; Human Genome Project; In Vitro; Knowledge; Left; Ligand Binding; Ligands; Link; Location; Measurement; Measures; Mediating; Monitor; Mutagenesis; N-terminal; Numbers; Oranges; Outcome; Positioning Attribute; Protein Binding; Protein Region; Proteins; Range; Relative (related person); Repression; Research Personnel; Role; Screening procedure; Sequence Analysis; Specificity; Structure; Tertiary Protein Structure; Testing; Transcriptional Regulation; Transplantation; Tweens; Variant; Work; design; dimer; ear helix; functional gain; gene repression; improved; in vivo; multidisciplinary; novel; pathogen; programs; research study; response

DESCRIPTION (provided by applicant): Protein homologues often have common functions, with variation evolving via amino acid substitutions at specificity determinants (SDs). These residues may be located in binding sites or other "long-range" regions of the protein. Predicting SDs is a current target of bioinformatics sequence analyses. Our long- term goal is to experimentally identify long-range SDs in the Lacl/GaIR family; the proposed work focuses on SDs in the linker that connects the DMA-binding domain to the regulatory domain. Hypotheses are derived from 4 different prediction strategies, which are only in partial agreement with each other. The common function of the Lacl/GaIR proteins - transcription control - allows "moderate through-put" assays of function so that all predictions may be compared. We will test hypotheses in multiple homologues to address the question: Do homologues utilize all SDs available to the common fold (a frequent assumption of prediction algorithms) or do different functions require only a subset of potential SDs? Further, the aspect of function affected by changing an SD cannot yet be reliably predicted, nor is it clear whether a functional change for one SD is the same in all homologues. Our experiments will monitor different aspects of Lacl/GaIR function, including DMA specificity, DNA affinity, and allosteric response to binding regulatory effector molecules. Proposed experiments utilize chimeras comprising the Lacl DMA-binding domain and regulatory domains from E. coli paralogues. Linkers come from Lacl or paralogues. Since each naturally-occurring Lacl/GaIR protein recognizes a different DNA ligand, the common DNA-binding domain allows us to more easily parse functional contributions from binding site and long-range SDs. By definition, making an amino acid substitu- tion at an SD will change function. We will use in vivo repression/response to effector and in vitro thermody- namic measurements of affinity/allosteric response to characterize the chimeras and potential SD variants. Specificity for alternative DNA ligands will be determined. Experiments are designed to answer the following questions: Aim 1: Can one linker facilitate allosteric communication with a variety of regulatory domains? [or do altered linker SD interactions abolish this function?] Aim 2: What are the functional contributions from specific positions in the linker? Aim 3: Can knowledge of SDs [in one linker] be used to transplant lacO1 DNA-binding to other linkers? Results will: (1) Identify the locations of SDs and determine if they make similar functional contributions to several homologues; (2) Yield a list of empirical rules for creating novel Lacl/GaIR proteins with biotechnological utility via domain recombination; and (3) Test the current prediction algorithms and generate a new sequence/function database for improving predictions. This work will facilitate expanded use of data generated by the Human Genome Project.

描述（由申请人提供）：蛋白质同源物通常具有共同的功能，并且在特异性决定因素（SDS）下通过氨基酸取代而变化。这些残基可能位于蛋白质的结合位点或其他“远程”区域。预测SDS是生物信息学序列分析的当前目标。我们的长期目标是在LACL/GAIR家族中实验识别长期SD；所提出的工作集中于将DMA结合域与调节域连接的接头中的SD。假设源自4种不同的预测策略，这些策略仅是部分一致的。 LAC/GAIR蛋白的常见功能 - 转录控制 - 允许对功能的“中等贯穿贯穿”测定法，以便可以比较所有预测。我们将在多个同源物中检验假设来解决以下问题：同源物是否利用可用的所有SD（经常假设预测算法），或者不同的功能仅需要一个潜在SDS的子集？此外，还不能可靠地预测受SD更改影响的函数方面，也不清楚一个SD的功能更改在所有同源物中是否相同。我们的实验将监测LAC/GAIR函数的不同方面，包括DMA特异性，DNA亲和力以及对结合调节效应分子的变构响应。提出的实验利用嵌合体，其中包括来自大肠杆菌旁程的LAC DMA结合结构域和调节域。链接器来自LACL或Paralogues。由于每个天然溶解的LACL/GAIR蛋白都识别出不同的DNA配体，因此常见的DNA结合结构域使我们可以从结合位点和远程SDS中更容易解析功能贡献。根据定义，在SD上进行氨基酸取代将改变功能。我们将在体内抑制/对效应子的反应和体外热力学测量亲和力/变构反应的测量，以表征嵌合体和潜在的SD变体。将确定替代DNA配体的特异性。实验旨在回答以下问题：目标1：一个接头可以促进与各种监管域的变构通信吗？ [或更改的链接器SD交互是否废除了此功能？] AIM 2：链接器中特定位置的功能贡献是什么？ AIM 3：SDS的知识能否用于将LACO1 DNA结合移植到其他接头？结果将：（1）确定SD的位置，并确定它们是否对几种同源物做出了相似的功能贡献； （2）产生通过域重组创建具有生物技术实用性的新型LACL/GAIR蛋白的经验规则列表； （3）测试当前的预测算法并生成一个新的序列/功能数据库，以改善预测。这项工作将促进人类基因组项目生成的数据的扩展使用。