Structural studies of transcriptional regulators

转录调节因子的结构研究

• 批准号: 7742148
• 负责人: MITCHELL LEWIS
• 金额: $ 31.66万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7742148
• 关键词: Animals; Binding; Biochemical; Biological Assay; Collaborations; DNA; DNA Binding Domain; DNA-Protein Interaction; Data; Development; Drops; Funding; Future; Genetic; Genetic Transcription; Grant; Housing; Lac Repressors; Libraries; Ligand Binding; Manuscripts; Modeling; Molecular; Molecular Biology; New England; Phenotype; Positioning Attribute; Proteins; Publications; Publishing; R-factor; Randomized; Reporting; Repressor Molecules; Research; Site; Structure; System; Techniques; Time; Writing; base; college; graduate student; human disease; in vivo; mutant; novel; promoter; resistance factors; skills; wasting

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）：本提案的总体目标是创建可以调节动物系统转录的转基因抑制因子。该授权描述了我们计划如何创建抑制因子，(a)可以识别不同的操作符，(b)可以用无毒诱导剂诱导。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lac阻遏物的结构研究，我们通过随机化与操作序列中的碱基进行特定相互作用的残基，创建了阻遏物分子的定向文库。我们将通过体内实验确定交替序列如何与特定碱基相互作用。这些修饰的阻遏物序列的多样性将用于揭示蛋白质- dna识别的分子基础。我们还建议创建异二聚体阻遏物，可以结合更多不同的操作符序列。大多数抑制子都能识别接近对称的操作符序列。异二聚体阻遏子具有识别具有两个不同半位点的操作子的能力，因此可以特异性地与几乎任何DNA结合。我们的最终目标是创造能够被现有的和新的诱导剂更有效地诱导的阻遏物。我们创建了一个突变体库，其中排列配体结合袋的残基是随机的。我们将在这个文库中寻找可诱导的突变抑制因子。然后，这些突变的抑制因子可以与突变的DNA结合域结合，产生与野生型lac抑制因子不同的新基因开关。在未来，一种改良的lac阻遏子可用于调控多种启动子。内源性基因座可以打开和关闭，以创建人类疾病和正常发育的可逆模型。