Functional effects of exchanging domains and linkers in transcription regulators

描述（由申请人提供）：蛋白质同源物通常具有共同的功能，其变异通过特异性决定因子（SDs）的氨基酸取代而进化。这些残基可能位于结合位点或蛋白质的其他“远程”区域。预测SDs是目前生物信息学序列分析的一个重要目标。我们的长期目标是通过实验鉴定Lacl/GaIR家族中的远程SDs；建议的工作重点是连接dma结合域和调控域的连接器中的SDs。假设来自4种不同的预测策略，它们之间只是部分一致。Lacl/GaIR蛋白的共同功能-转录控制-允许“中等吞吐量”的功能分析，以便所有预测可以进行比较。我们将在多个同系物中测试假设，以解决以下问题：同系物是否利用所有可用于共同折叠的SDs（预测算法的常见假设），或者不同的函数是否只需要潜在SDs的子集？此外，改变SD对功能的影响还不能可靠地预测，也不清楚一个SD的功能改变在所有同源物中是否相同。我们的实验将监测Lacl/GaIR功能的不同方面，包括DMA特异性、DNA亲和力和对结合调节效应分子的变构反应。拟议的实验利用嵌合体包括Lacl dma结合结构域和大肠杆菌旁系的调控结构域。链接器来自Lacl或paralogues。由于每个天然存在的Lacl/GaIR蛋白识别不同的DNA配体，共同的DNA结合结构域使我们更容易解析结合位点和远程SDs的功能贡献。根据定义，在SD上进行氨基酸取代将改变其功能。我们将使用体内抑制/对效应物的反应和体外亲和/变构反应的热力学测量来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：目的1：一个连接体能否促进与各种调控域的变构通信？或者改变的链接器SD相互作用取消了这个功能？目标2：链接器中特定位置的功能贡献是什么？目标3：是否可以将SDs（在一个连接体中）的知识用于移植lacO1 dna结合到其他连接体上？结果将：(1)确定SDs的位置，并确定它们是否对几个同源物具有相似的功能贡献；(2)通过结构域重组产生具有生物技术实用性的新型Lacl/GaIR蛋白的经验规则列表；(3)对现有的预测算法进行测试，生成新的序列/函数数据库以改进预测。这项工作将促进人类基因组计划产生的数据的扩大使用。

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.