Functional effects of exchanging domains and linkers in transcription regulators

描述(由申请人提供)：蛋白质同系物通常具有共同的功能，变异是通过特异性决定因素(SDS)上的氨基酸替换而演变的。这些残基可能位于蛋白质的结合部位或其他“长程”区域。预测十二烷基硫酸酯是当前生物信息学序列分析的目标。我们的长期目标是通过实验确定Lacl/Gair家族中的长距离SDS；拟议的工作重点是连接DMA结合结构域和调节域的连接子中的SDS。假设是从4种不同的预测策略得出的，它们之间只有部分一致。Lacl/Gair蛋白的共同功能--转录控制--允许对功能进行“适度的吞吐量”分析，以便可以比较所有的预测。我们将测试多个同源序列中的假设来解决这个问题：同源序列是否利用了公共折叠可用的所有SD(预测算法的一个常见假设)，或者不同的功能是否只需要潜在SD的子集？此外，改变SD影响的功能方面还不能可靠地预测，也不清楚一个SD的功能改变是否在所有同源物中都是相同的。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。拟议的实验利用了由Lacl DMA结合结构域和来自E.Coli类似物的调节域组成的嵌合体。链接器来自Lacl或Paralog。由于每个自然产生的Lacl/Gair蛋白识别不同的DNA配体，共同的DNA结合域使我们能够更容易地从结合位点和长程SDS中解析功能贡献。根据定义，在SD中进行氨基酸替代将改变功能。我们将使用体内对效应器的抑制/反应和体外亲和力/变构反应的热力学测量来表征嵌合体和潜在的SD变体。替代DNA配体的特异性将被确定。实验旨在回答以下问题：目标1：一个连接子能促进变构与各种调控结构域的通讯吗？[或者，改变链接器SD的相互作用会取消这一功能吗？]目标2：连接子中特定位置的功能贡献是什么？目的3：了解SDS[在一个连接子中]是否可以用于将lacO1DNA结合移植到其他连接子上？结果将：(1)确定SDS的位置，并确定它们是否对几个同源物做出类似的功能贡献；(2)产生一系列经验规则，用于通过结构域重组创建具有生物技术实用价值的新Lacl/Gair蛋白质；以及(3)测试当前的预测算法，并生成新的序列/功能数据库以改进预测。这项工作将有助于扩大人类基因组计划产生的数据的使用。

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.