Structural studies of transcriptional regulators

转录调节因子的结构研究

• 批准号: 7932666
• 负责人: MITCHELL LEWIS
• 金额: $ 17.65万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7932666
• 关键词: Amino Acids; Animals; Architecture; Binding; Biological Assay; Biological Models; Breast Cancer Cell; Cancer cell line; DNA; DNA Binding Domain; DNA Sequence; DNA-Protein Interaction; Data; Databases; Development; Funding; Future; Gene Expression; Genetic; Genetic Transcription; Goals; Grant; Lac Operon; Lactose; Libraries; Ligand Binding; Mammals; Modeling; Molecular; Plants; Process; Proteins; Randomized; Repressor Molecules; Research; Signal Transduction; Site; Specificity; Stem cells; System; Transcriptional Regulation; base; gene therapy; high throughput screening; human disease; in vivo; monomer; mutant; novel; promoter; protein expression; success; three dimensional structure

DESCRIPTION (provided by applicant): The overall aim of this proposal is to create genetically altered repressors that can regulate transcription in animal systems. This grant describes how we plan to create repressors that (a) can recognize different operators and (b) can be induced with non-toxic inducers. The research proposed will also help to elucidate principles governing protein-DNA interactions. Building on our structural studies of the lac repressor, we have created directed libraries of repressor molecules by randomizing the residues making specific interactions with bases in the operator sequence. We will determine how alternate sequences can interact with specific bases using an in vivo assay. The diversity in the sequences of these modified repressors will be used to unravel the molecular basis for protein-DNA recognition. We also propose to create heterodimeric repressor that can bind more diverse operator sequences. Most repressors recognize operator sequences that are nearly symmetric. A heterodimeric repressor has the ability to recognize an operator with two distinct half sites and therefore bind specifically to virtually any DNA. Our final aim is to create repressors that can be induced more efficiently by both existing and novel inducers. We have created a library of mutants where the residues lining the ligand binding pocket have been randomized. We will search this library for mutant repressors that are inducible. These mutant repressors can then be combined with mutant DNA binding domains to create new genetic switches distinct from the wild-type lac repressor. In the future a modified lac repressor could be used to regulate a variety of promoters. Endogenous loci could be switched on and off to create reversible models of human disease and normal development.

描述（由申请人提供）：该提案的总体目标是创建可以调节动物系统中转录的遗传改变的阻遏物。这项资助描述了我们计划如何创建阻遏物，（a）可以识别不同的运营商和（B）可以诱导与无毒诱导剂。这项研究还将有助于阐明蛋白质-DNA相互作用的原理。基于我们对乳糖阻遏物的结构研究，我们通过随机化与操纵子序列中的碱基发生特异性相互作用的残基来创建阻遏物分子的定向文库。我们将确定如何交替序列可以使用体内测定与特定的碱基相互作用。这些修饰阻遏物序列的多样性将被用来解开蛋白质-DNA识别的分子基础。我们还建议创建异源二聚体阻遏物，可以结合更多样化的操纵子序列。大多数阻遏物识别几乎对称的操纵子序列。异源二聚体阻遏物具有识别具有两个不同半位点的操纵子的能力，因此特异性结合几乎任何DNA。我们的最终目标是创造一种可以被现有的和新的诱导剂更有效地诱导的阻遏物。我们已经创建了突变体库，其中排列配体结合口袋的残基已经被随机化。我们将在这个文库中寻找可诱导的突变阻遏物。然后，这些突变阻遏物可以与突变DNA结合结构域组合，以产生不同于野生型乳糖阻遏物的新的遗传开关。在未来，一种经过修饰的乳糖阻遏物可以用来调控多种启动子。内源性基因座可以被打开和关闭，以创建人类疾病和正常发育的可逆模型。