PHYSICAL AND GENETIC STUDIES OF REGULATORY PROTEINS

调节蛋白的物理和遗传学研究

• 批准号: 2173933
• 负责人: KATHLEEN S MATTHEWS
• 金额: $ 30.15万
• 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/2173933
• 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.

调控转录启动的基因调控过程依赖于 原核生物和真核生物中特异的蛋白质-DNA相互作用 有机体。大肠埃希菌中的原型负转录调控因子 Coli是lac抑制蛋白，它阻止了mRNA合成的启动。 用于乳糖代谢酶，除非乳糖在 环境，和色氨酸抑制物，它抑制几个基因的表达 不同的系统对细胞内色氨酸水平的反应。这个 同源异型超双胸蛋白用于确定节段身份 通过改变特定基因的转录在果蝇中的发育。 这些蛋白质识别多个特定的DNA序列来执行它们的 监管职能。乳胶抑制子通过它的两个 操纵子位点/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。低聚物 紫胶界面的形成、亚基组装及特征 我们将使用各种基因、化学物质来探索阻遏因子。 和物理方法。由Trp结合的整个序列曲目 阻遏蛋白和超生物胸腔蛋白将通过从 用聚合酶链法扩增的随机寡核苷酸混合物 反应和测序。为了阐明绑定的模式， 蛋白质的化学计量：与寡核苷酸形成的络合物中的DNA 和多位点DNA将被评估，环境的影响 将测量有关绑定的条件。色氨酸抑制因子的证据 四聚体形成和超双胸蛋白二聚体形成提示 结合串联位点和/或DNA环的协作性潜力 通过遥远的地点形成；这些令人兴奋的可能性将被探索 使用不同间距和不同的多点DNA构建物 超级卷曲。阐明超级胸腔积液形成环路的可能性 行动可以提供对这一机制的一个方面的洞察 蛋白质作为多个识别位点发挥其发育控制作用 在一个受调控的转录单位内的基因已经被确定。头寸 超强胸膜蛋白与其分离蛋白之间的紧密相互作用 同源结构域和DNA靶点将通过DNA修饰来探索。 将采用蛋白质分解和蛋白质片段的细菌表达。 探索超生物胸膜蛋白的结构域结构，并 确定与二聚体形成有关的位点，并确认DNA 结合部位。这些研究的结果将显著扩大。 我们对这些基本基因的结构和功能的理解 调节蛋白。