PHYSICAL AND GENETIC STUDIES OF REGULATORY PROTEINS

调节蛋白的物理和遗传学研究

• 批准号: 3271143
• 负责人: KATHLEEN S MATTHEWS
• 金额: $ 27.36万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271143
• 关键词: DNA; DNA binding protein; DNA footprinting; DNA methylation; bacterial genetics; chemical association; chemical binding; conformation; dimer; fluorescence spectrometry; gel mobility shift assay; gene induction /repression; genetic operator element; homeobox genes; ionic strengths; lac operon; mutant; nucleic acid sequence; nucleic acid structure; polymerase chain reaction; protein engineering; protein sequence; protein structure function; site directed mutagenesis; stoichiometry; temperature; transcription factor; tryptophan

Genetic regulatory processes that modulate transcription initiation rely on specific protein-DNA interactions in both prokaryotic and eukaryotic organisms. Prototypic negative transcriptional regulators in Escherichia coli are lac repressor protein, which prevents initiation of mRNA synthesis for the lactose metabolic enzymes unless lactose is available in the environment, and trp repressor, which represses expression of several different systems in response to intracellular tryptophan levels. The homeotic Ultrabithorax protein serves to specify segmental identity during development in Drosophila by altering transcription of specific genes. These proteins recognize multiple, specific DNA sequences to perform their regulatory function. The lac repressor forms looped DNAs via its two operator sites/tetramer. These loops require tetramer, as dimeric repressor proteins that cannot form loops have been generated. Oligomer formation, subunit assembly and character of the interfaces between lac repressor protomers will be explored using a variety of genetic, chemical and physical methods. The entire repertoire of sequences bound by trp repressor and Ultrabithorax protein will be identified by selection from a random mixture of oligonucleotides, amplification by polymerase chain reaction, and sequencing. To clarify the mode of binding, the stoichiometry of protein:DNA in the complexes formed with oligonucleotides and multi-site DNAs will be evaluated, and the effects of environmental conditions on binding will be measured. Evidence for trp repressor tetramer formation and for Ultrabithorax protein dimer formation suggests the potential for cooperativity in binding tandem sites and/or DNA loop formation via distant sites; these exciting possibilities will be explored using various multi-site DNA constructs with differences in spacing and supercoiling. Clarifying the potential for loop formation in Ultrabithorax action may provide insight into one aspect of the mechanism by which this protein exerts its developmental control, as multiple recognition sites within a regulated transcription unit have been identified. The positions of close interactions between Ultrabithorax protein or its isolated homeodomain and DNA target sites will be explored by DNA modification. Proteolysis and bacterial expression of protein fragments will be employed to explore the domain structure of the Ultrabithorax protein and to identify sites involved in dimer formation as well as to confirm DNA binding sites. The results from these studies will significantly expand our understanding of the structure and function of these essential genetic regulatory proteins.

调节转录起始的遗传调控过程依赖于 原核和真核生物中特异性蛋白质-DNA相互作用 有机体 大肠杆菌中原型负转录调控因子的研究 大肠杆菌是lac阻遏蛋白，其阻止mRNA合成的起始 对于乳糖代谢酶，除非乳糖在 环境和色氨酸阻遏物，其抑制几种 不同系统对细胞内色氨酸水平的反应。 的 同源异型的超双胸蛋白质用于指定片段身份， 通过改变特定基因的转录来促进果蝇的发育。 这些蛋白质识别多个特定的DNA序列，以执行其功能。 调节功能。 lac阻遏物通过其两个 操作位点/四聚体。 这些环需要四聚体，如二聚体 已经产生了不能形成环的阻遏蛋白。 低聚 紫胶的形成、亚基组装及界面特征 阻遏物原体将使用各种遗传、化学和生物技术来探索。 物理方法。 由trp结合的整个序列库 阻遏物和Ultrabithorax蛋白将通过从一个 寡核苷酸随机混合物，聚合酶链扩增 反应和测序。 为了阐明绑定的模式， 与寡核苷酸形成的复合物中蛋白质：DNA的化学计量 和多个地点的DNA将进行评估，和环境的影响， 将测量结合的条件。 trp阻遏物的证据 四聚体形成和超双胸蛋白二聚体形成表明 结合串联位点和/或DNA环中协同性潜力 通过遥远的网站形成;这些令人兴奋的可能性将被探索 使用具有不同间距的各种多位点DNA构建体， 超螺旋 阐明在超双胸中形成袢的可能性 行动可以提供洞察机制的一个方面， 蛋白质发挥其发育控制，作为多个识别位点， 在一个受调控的转录单位内已经被鉴定。 的位置 Ultrabithorax蛋白或其分离的 同源结构域和DNA靶位点将通过DNA修饰来探索。 将采用蛋白水解和蛋白片段的细菌表达 探索Ultrabithorax蛋白的结构域结构， 鉴定参与二聚体形成的位点以及确认DNA 结合位点。 这些研究的结果将大大扩展 我们对这些重要基因的结构和功能的理解 调节蛋白